Nucleotide and amino acid sequences of canine herpesvirus GD and uses therefor

ABSTRACT

Disclosed and claimed are nucleotides for genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues. These genes encode polypeptides of 879, 459 and 345 amino acids, respectively, which are also disclosed and claimed. The genes are useful as DNA probes or, for preparing PCR primers. The polypeptides are useful in antigenic, immunological or vaccine compositions. The nucleotides can be expressed in any suitable vector system, allowing for production of the polypeptides. Additionally, the vector system containing any or any combination of the nucleotides can be employed in an antigenic, immunological or vaccine composition, such as a poxvirus vector system, e.g., a CHV-vaccinia or avipox virus recombinant, as can the products from expression, i.e., the gB, gC and gD glycoproteins. Antibodies elicited by the glycoproteins or from expression of the vector containing the nucleotide(s) are also useful. Methods for making and using the composition are also disclosed and claimed. Also, specific canarypox-CHV gB, gC and gD recombinants vCP 320, vCP322 and vCP294 and methods for making and using them are also disclosed and claimed.

RELATED APPLICATIONS

This application is a division of application Ser. No. 08/413,118, filedMar. 29, 1995, now U.S. Pat. No. 5,688,920, which is a continuation inpart of application Ser. No. 08/220,151, filed Mar. 30, 1994, now U.S.Pat. No. 5,529,780.

Each of the above-mentioned patents are hereby incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to canine herpesvirus (CHV), nucleotides orisolated nucleic acids encoding the CHV gB, gC and gD glycoproteins, andthe amino acid sequences thereof, vectors, such as a recombinantpoxvirus, e.g., vaccinia and avipox virus recombinants, containing theCHV gB, gC and/or gD coding or expressing the same, glycoproteinstherefrom, vaccines, immunological or antigenic compositions from thenucleotide (such as from vectors, for instance, recombinant poxvirus,e.g., vaccinia or avipox virus recombinants containing the CHV, gB, gCand/or gD coding and expressing glycoprotein(s) therefrom), or, from theglycoproteins, for instance, from expression of the nucleotides in avector system, and, to methods employing the nucleotides, glycoproteins,and compositions.

Several publications are cited in the following text, with full citationof each set forth in the section headed References. The publicationscited throughout the text are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Canine herpesvirus (CHV) causes a fatal, hemorrhagic disease in neonatalpuppies and a self-limiting, usually subclinical, upper respiratorytract infection in adult dogs (Appel, 1987). Little is known about thegenomic structure of CHV. The genome has not been mapped and nonucleotide sequence has been published. In particular, genes encodingimmunologically pertinent proteins have not been identified.

Herpesvirus glycoproteins mediate essential viral functions such ascellular attachment and penetration, cell to cell spread of the virusand, importantly, determine the pathogenicity profile of infection.Herpesvirus glycoproteins are critical components in the interactionwith the host immune system (Spear, 1985a; Spear 1985b). Herpesvirusglycoproteins are antigens recognized by both the humoral and cellularimmune systems and, have been shown to evoke protective immune responsesin vaccinated hosts (Wachsman et al., 1987; Marchioli et al., 1987;Eberle et al., 1980; Papp-Vid et al., 1979).

During a herpesvirus infection, the majority of the immune response isdirected against viral envelope glycoproteins. These antigens have beenshown to elicit both humoral and cellular immune responses. Severalreports have indicated that in other herpesvirus systems immunizationwith the herpesvirus gB, gC and/or gD glycoproteins can induce aprotective immune response.

The well characterized glycoproteins of herpes simplex virus include gB,gC, gD, gE, gG, gH, gI, gJ, gK, gL and gM (Spear, 1985a; Spear 1985b;Ackermann et al., 1986; Frink et al. 1983; Frame et al., 1986;Longnecker et al., 1987; Richman et al., 1986; Swain et al., 1985;Zezulak, 1984; Roizman and Sears, 1990; Hutchinson et al., 1992a;Hutchinson et al., 1992b; Baines and Roizman, 1993). A number of studieshave indicated the importance of herpes simplex virus glycoproteins ineliciting immune responses. Hence, it has been reported that gB and gDcan elicit important immune responses (Berman et al., 1983; Cantin etal., 1987; Cremer et al., 1985; Lasky et al., 1984; Martin et al.,1987a; Martin et al., 1987b; Paoletti et al., 1984; Perkus et al., 1985;Rooney et al., 1988; Wachsman et al., 1987; Zarling et al., 1986a;Zarling et al., 1986b). gC can stimulate class I restricted cytotoxiclymphocytes (Glorioso et al., 1985; Rosenthal et al., 1987) whereas gDcan stimulate class II cytotoxic T cell responses (Martin et al., 1987a;Martin et al,. 1987b; Wachsman et al., 1987; Zarling et al., 1986a;Zarling 1986b). gG was shown to be a target for complement-dependentantibody directed virus neutralization (Sullivan et al., 1987; Sullivanet al., 1988). A number of glycoproteins from other herpesviruses havealso been shown to elicit important immune responses.

Both subtypes of equine herpesvirus (EHV) express six abundantglycoproteins (Allen et al., 1986; Allen et al., 1987). The genomicportions of the DNA sequences encoding gp2, gp10, gp13, gp14, gp17/18,and gp21/22a have been determined using lambda gtll expression vectorsand monoclonal antibodies (Allen et al., 1987). Glycoproteins gp13 andgp14 were located in the same locations within the L component of thegenome to which the gC and gB homologs, respectively, of herpes simplexvirus map (Allen et al., 1987). The envelope glycoproteins are theprincipal immunogens of herpesviruses involved in eliciting both humoraland cellular host immune responses (Ben-Porat et al., 1986; Cantin etal., 1987; Glorioso et al., 1984; Wachsman et al., 1988; Wachsman etal., 1989) and so are of the highest interest for those attempting todesign vaccines.

Recently, the nucleotide sequence of the Kentucky T431 strain of theEHV-1 transcriptional unit encoding gp13 has been reported (Allen etal., 1988). The glycoprotein was shown to be homologous to the herpessimplex virus (HSV) gC-1 and gC-2, to the pseudorabies virus (PRV) gIIIand the varicella-zoster virus (VZV) gpV (Allen et al., 1988). EHV-1gp13 is thus the structural homolog of the herpesvirus gC-likeglycoproteins.

The nucleotide sequence of EHV-1 gp14 (Whalley et al., 1989; Riggio etal., 1989) has recently been reported. Analysis of the predicted aminoacid sequence of gp14 glycoprotein revealed significant homology to thecorresponding glycoprotein of HSV, gB.

Monoclonal antibodies directed against some EHV-1 glycoproteins havebeen shown to be neutralizing (Sinclair et al., 1989). Passiveimmunization experiments demonstrated that monoclonal antibodiesdirected against gp13 or gp14 (Shimizu et al., 1989) or against gp13,gp14 or gp17/18 (Stokes et al., 1989) could protect hamsters against alethal challenge. Other gB and gC glycoprotein analogs are also involvedin protection against diseases caused by alphaherpesviruses (Cantin etal., 1987; Cranage et al., 1986; Glorioso et al., 1984).

Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent ofAujesky's disease. The PRV genome consists of a 90×10⁶ dalton doublestranded DNA (Rubenstein et al., 1975) separated by inverted repeatsequences into unique long (U_(L)) or unique short (U_(S)) segments(Stevely, 1977; Ben-Porat et al., 1979). The PRV genome encodesapproximately 100 polypeptides whose expression is regulated in acascade-like fashion similar to other herpesviruses (Ben-Porat et al.,1985; Hampl et al., 1984).

PRV glycoprotein gp50 is the Herpes simplex virus type 1 (HSV-1) gDanalog (Wathen et al., 1984). The DNA open reading frame encodes 402amino acids (Petrovskis et al., 1986). The mature glycosylated form(50-60 kDa) contains O-linked carbohydrate without N-linkedglycosylation (Petrovskis et al., 1986). Swine serum is highly reactivewith PRV gp50, suggesting its importance as an immunogen. Monoclonalantibodies to gp50 neutralize PRV in vitro with or without complement(Wathen et al., 1984; Wathen 1985; Eloit et al., 1988) and passivelyprotect mice (Marchioli et al., 1988; Wathen et al., 1985; Eloit et al.,1988) and swine (Marchioli et al., 1988). Vaccinia virus recombinantsexpressing PRV gp50 induced serum neutralizing antibodies and protectedboth mice and swine against lethal PRV challenge (Kost et al., 1989;Marchioli et al., 1987; Ishii et al., 1988).

PRV gIII is the HSV-1 gC analog (Robbins et al., 1986). Functionalreplacement of PRV gIII by HSVgC was not observed (Whealy et al., 1989).Although PRV gIII is nonessential for replication in vitro (Wathen etal., 1986; Robbins et al., 1986), the mature glycosylated form (98 kDa)is an abundant constituent of the PRV envelope. Anti-gpIII monoclonalantibodies neutralize the virus in vitro with or without complement(Hampl et al., 1984; Eloit et al., 1988; Wathen et al., 1986) and canpassively protect mice and swine (Marchioli et al., 1988). The PRVglycoprotein gIII can protect mice and swine from lethal PRV challengeafter immunization with a Cro/gIII fusion protein expressed in E. coli(Robbins, A., R. Watson, L. Enquist, European Patent application0162738A1) or when expressed in a vaccinia recombinant (Panicali, D., L.Gritz, G. Mazzara, European Patent application 0261940A2).

PRV gpII is the HSV-1 gB homolog (Robbins et al., 1987). Monoclonalantibodies directed against PRV gpII have been shown to neutralize thevirus in vitro (Ben-Porat et al., 1986) with or without complement(Wittmann et al., 1989). Moreover, passive immunization studiesdemonstrated that neutralizing monoclonal antibodies partially protectedswine (Marchioli et al., 1988). Immunization with NYVAC (highlyattenuated vaccinia virus)-based recombinants expressing pseudorabiesvirus (PRV) gII (gB) or gp50 (gD) has been shown to protect swineagainst a virulent PRV challenge (Brockmeier et al., 1993). Furthermore,vaccinia recombinants expressing PRV gII and gp50, or gII, gIII (gC) andgp50 have been shown to elicit a higher level of protection thanrecombinants expressing gII or gp50 alone, suggesting a potentialsynergistic effect with these glycoproteins (Riviere et al., 1992).

The herpes simplex virus type 1 (HSV1) genome encodes at least elevenantigenically distinct glycoproteins: gB, gC, gD, gE, gG, gH, gI, gJ,gK, gL and gM (Roizman et al., 1990). Mice immunized with purified HSV1gB, gC or gD are protected against lethal HSV1 challenge (Chan, 1983).Mice have also been protected against lethal HSV1 or HSV2 challenge bypassive immunization with antibodies to total HSV1 (Davis et al., 1979)or HSV2 (Oakes et al., 1978) virus and with antibodies to the individualHSV2 gB, gC, gD or gE glycoproteins (Balachandran et al., 1982).

Vaccinia virus vectors expressing HSV1 gB (McLaughlin-Taylor et al.,1988) and HSV1 gC (Rosenthal et al., 1987) have been shown to inducecytotoxic T-cell responses. In addition, it has been shown that miceimmunized with recombinant vaccinia virus expressing either HSV1 gB(Cantin et al., 1987), HSV1 gC (Weir et al., 1989) or HSV1 gD (Paolettiet al., 1984) are protected against a lethal challenge of HSV1. Arecombinant vaccinia virus expressing HSV1 gD has also been shown to beprotective against HSV2 in a guinea pig model system (Wachsman et al.,1987).

Bovine herpesvirus 1 (BHV1) specifies more than 30 structuralpolypeptides, 11 of which are glycosylated (Misra et al., 1981). Threeof these glycoproteins, gI, gIII and gIV, have been characterized andfound to be homologous to the herpes simplex virus (HSV) glycoproteinsgB, gC and gD (Lawrence et al., 1986; Zamb, 1987). Immunization withpurified bovine herpesvirus type 1 (BHV1) gI (gB), gIII (gC) and/or gIV(gD) has been shown to protect cattle against a BHV1/Pasteurellahaemolytica challenge (Babiuk et al., 1987).

Feline herpesvirus type-1 (FHV-1) has been shown to contain at least 23different proteins (Meas et al., 1984; Fargeaud et al., 1984). Of these,at least five are glycosylated (Fargeaud et al., 1984; Compton, 1989)with reported molecular masses ranging from 120 kDa to 60 kDa. The FHV-1glycoproteins have been shown to be immunogenic (Meas et al., 1984;Compton, 1989). Like several other alphaherpesviruses, FHV-1 appears tohave a homolog of glycoprotein B (gB) of HSV-1 (Maeda et al., 1992). TheFHV-1 gB glycoprotein is a 134 kDa complex which is dissociated withB-mercaptoethanol into two glycoproteins of 66 kDa and 60 kDa. The FHV-1DNA genome is approximately 134 Kb in size (Rota et al., 1986).

Epstein Barr Virus (EBV), a human B lymphotropic herpesvirus, is amember of the genus lymphocryptovirus which belongs to the subfamilygammaherpesvirus (Roizman et al., 1990). Since the EBV genome wascompletely sequenced (Baer et al., 1984) as the genomes of VZV (Davisonet al., 1986), HSV1 (McGeoch et al., 1988), MCHV (Chee et al., 1990) andEHV1 (Telford et al., 1992) numerous homologies between these differentherpesviruses have been described (Kieff et al., 1990).

Human cytomegalovirus (HCMV) is a member of the betaherpesvirinaesubfamily (family Herpesviridae). Three immunologically distinctfamilies of glycoproteins associated with the HCMV envelope have beendescribed (Gretch et al., 1988): gCI (gp55 and gp93-130); gCII(gp47-52); and gCIII (gp85-p145). The gene coding for gCI is homologousto HSVI gB.

In addition, immunization with a fowlpox recombinant expressing Marek'sdisease virus (MDV) gB has been shown to protect chickens against avirulent MDV challenge (Nazarian et al., 1992).

The results of these studies indicate that an immune response againstgB, gC and/or gD glycoproteins can protect target species animalsagainst a herpesvirus challenge and, that the provision of nucleotidesfor CHV gB, gC and gD glycoproteins is a valuable advance over thecurrent state of the art as it allows for the provision of theglycoproteins and, antigenic, immunological or vaccine compositions fromthe vector systems or from the glycoproteins. Further, the glycoproteinsfrom expression of the nucleotides can be used to elicit antibodieswhich can be further used in antibody binding diagnostic assays, kits ortests for ascertaining the presence or absence in a sample such as seraof the glycoprotein(s) and therefore the presence or absence of CHV orof an immune or antigenic response (to either CHV or to theglycoproteins). Thus, many utilities flow from the provision of thenucleotides for CHV gB, gC and gD glycoproteins.

Various vector systems exist for the expression of exogenous DNA, suchas the phage, e.g., lambda, and E. coli systems (Allen et al., 1987;Robbins, EPA 0162738A1; Panicali, EPA 0261940A2, each of which isexpressly incorporated herein by reference).

Vaccinia virus and more recently other poxviruses have been used for theinsertion and expression of foreign genes. The basic technique ofinserting foreign genes into live infectious poxvirus involvesrecombination between pox DNA sequences flanking a foreign geneticelement in a donor plasmid and homologous sequences present in therescuing poxvirus (Piccini et al., 1987).

Specifically, the recombinant poxviruses are constructed in two stepsknown in the art and analogous to the methods for creating syntheticrecombinants of poxviruses such as the vaccinia virus and avipox virusdescribed in U.S. Pat. Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587,and 5,179,993, the disclosures of which are incorporated herein byreference.

First, the DNA gene sequence to be inserted into the virus, particularlyan open reading frame from a non-pox source, is placed into an E. coliplasmid construct into which DNA homologous to a section of DNA of thepoxvirus has been inserted. Separately, the DNA gene sequence to beinserted is ligated to a promoter. The promoter-gene linkage ispositioned in the plasmid construct so that the promoter-gene linkage isflanked on both ends by DNA homologous to a DNA sequence flanking aregion of pox DNA containing a nonessential locus. The resulting plasmidconstruct is then amplified by growth within E. coli bacteria (Clewell,1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).

Second, the isolated plasmid containing the DNA gene sequence to beinserted is transfected into a cell culture, e.g. chick embryofibroblasts, along with the poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively gives apoxvirus modified by the presence, in a nonessential region of itsgenome, of foreign DNA sequences. The term "foreign" DNA designatesexogenous DNA, particularly DNA from a non-pox source, that codes forgene products not ordinarily produced by the genome into which theexogenous DNA is placed.

Genetic recombination is in general the exchange of homologous sectionsof DNA between two strands of DNA. In certain viruses RNA may replaceDNA. Homologous sections of nucleic acid are sections of nucleic acid(DNA or RNA) which have the same sequence of nucleotide bases.

Genetic recombination may take place naturally during the replication ormanufacture of new viral genomes within the infected host cell. Thus,genetic recombination between viral genes may occur during the viralreplication cycle that takes place in a host cell which is co-infectedwith two or more different viruses or other genetic constructs. Asection of DNA from a first genome is used interchangeably inconstructing the section of the genome of a second co-infecting virus inwhich the DNA is homologous with that of the first viral genome.

However, recombination can also take place between sections of DNA indifferent genomes that are not perfectly homologous. If one such sectionis from a first genome homologous with a section of another genomeexcept for the presence within the first section of, for example, agenetic marker or a gene coding for an antigenic determinant insertedinto a portion of the homologous DNA, recombination can still take placeand the products of that recombination are then detectable by thepresence of that genetic marker or gene in the recombinant viral genome.Additional strategies have recently been reported for generatingrecombinant vaccinia virus.

Successful expression of the inserted DNA genetic sequence by themodified infectious virus requires two conditions. First, the insertionmust be into a nonessential region of the virus in order that themodified virus remain viable. The second condition for expression ofinserted DNA is the presence of a promoter in the proper relationship tothe inserted DNA. The promoter must be placed so that it is locatedupstream from the DNA sequence to be expressed.

Vaccinia virus has been used successfully to immunize against smallpox,culminating in the worldwide eradication of smallpox in 1980. In thecourse of its history, many strains of vaccinia have arisen. Thesedifferent strains demonstrate varying immunogenicity and are implicatedto varying degrees with potential complications, the most serious ofwhich are post-vaccinial encephalitis and generalized vaccinia(Behbehani, 1983).

With the eradication of smallpox, a new role for vaccinia becameimportant, that of a genetically engineered vector for the expression offoreign genes. Genes encoding a vast number of heterologous antigenshave been expressed in vaccinia, often resulting in protective immunityagainst challenge by the corresponding pathogen (reviewed in Tartagliaet al., 1990a).

The genetic background of the vaccinia vector has been shown to affectthe protective efficacy of the expressed foreign immunogen. For example,expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strainof vaccinia virus did not protect cottontop tamarins against EBV virusinduced lymphoma, while expression of the same gene in the WR laboratorystrain of vaccinia virus was protective (Morgan et al., 1988).

A fine balance between the efficacy and the safety of a vacciniavirus-based recombinant vaccine candidate is extremely important. Therecombinant virus must present the immunogen(s) in a manner that elicitsa protective immune response in the vaccinated animal but lacks anysignificant pathogenic properties. Therefore attenuation of the vectorstrain would be a highly desirable advance over the current state oftechnology.

A number of vaccinia genes have been identified which are non-essentialfor growth of the virus in tissue culture and whose deletion orinactivation reduces virulence in a variety of animal systems.

The gene encoding the vaccinia virus thymidine kinase (TK) has beenmapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir etal., 1983). Inactivation or complete deletion of the thymidine kinasegene does not prevent growth of vaccinia virus in a wide variety ofcells in tissue culture. TK⁻ vaccinia virus is also capable ofreplication in vivo at the site of inoculation in a variety of hosts bya variety of routes.

It has been shown for herpes simplex virus type 2 that intravaginalinoculation of guinea pigs with TK⁻ virus resulted in significantlylower virus titers in the spinal cord than did inoculation with TK⁺virus (Stanberry et al., 1985). It has been demonstrated thatherpesvirus encoded TK activity in vitro was not important for virusgrowth in actively metabolizing cells, but was required for virus growthin quiescent cells (Jamieson et al., 1974).

Attenuation of TK⁻ vaccinia has been shown in mice inoculated by theintracerebral and intraperitoneal routes (Buller et al., 1985).Attenuation was observed both for the WR neurovirulent laboratory strainand for the Wyeth vaccine strain. In mice inoculated by the intradermalroute, TK⁻ recombinant vaccinia generated equivalent anti-vaccinianeutralizing antibodies as compared with the parental TK⁺ vacciniavirus, indicating that in this test system the loss of TK function doesnot significantly decrease immunogenicity of the vaccinia virus vector.Following intranasal inoculation of mice with TK⁻ and TK⁺ recombinantvaccinia virus (WR strain), significantly less dissemination of virus toother locations, including the brain, has been found (Taylor et al.,1991a).

Another enzyme involved with nucleotide metabolism is ribonucleotidereductase. Loss of virally encoded ribonucleotide reductase activity inherpes simplex virus (HSV) by deletion of the gene encoding the largesubunit was shown to have no effect on viral growth and DNA synthesis individing cells in vitro, but severely compromised the ability of thevirus to grow on serum starved cells (Goldstein et al., 1988). Using amouse model for acute HSV infection of the eye and reactivatable latentinfection in the trigeminal ganglia, reduced virulence was demonstratedfor HSV deleted of the large subunit of ribonucleotide reductase,compared to the virulence exhibited by wild type HSV (Jacobson et al.,1989).

Both the small (Slabaugh et al., 1988) and large (Schmitt et al., 1988)subunits of ribonucleotide reductase have been identified in vacciniavirus. Insertional inactivation of the large subunit of ribonucleotidereductase in the WR strain of vaccinia virus leads to attenuation of thevirus as measured by intracranial inoculation of mice (Child et al.,1990).

The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced(Shida, 1986). The HA gene of vaccinia virus is nonessential for growthin tissue culture (Ichihashi et al., 1971). Inactivation of the HA geneof vaccinia virus results in reduced neurovirulence in rabbitsinoculated by the intracranial route and smaller lesions in rabbits atthe site of intradermal inoculation (Shida et al., 1988). The HA locuswas used for the insertion of foreign genes in the WR strain (Shida etal., 1987), derivatives of the Lister strain (Shida et al., 1988) andthe Copenhagen strain (Guo et al., 1989) of vaccinia virus. RecombinantHA⁻ vaccinia virus expressing foreign genes have been shown to beimmunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988;Shida et al., 1987) and protective against challenge by the relevantpathogen (Guo et al., 1989; Shida et al., 1987).

Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks onthe chorioallantoic membrane of chicken eggs. Spontaneous deletionswithin the cowpox genome generate mutants which produce white pocks(Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDaprotein encoded by an early gene (Pickup et al., 1986). This gene, whichhas homology to serine protease inhibitors, has been shown to inhibitthe host inflammatory response to cowpox virus (Palumbo et al., 1989)and is an inhibitor of blood coagulation.

The u gene is present in WR strain of vaccinia virus (Kotwal et al.,1989b). Mice inoculated with a WR vaccinia virus recombinant in whichthe u region has been inactivated by insertion of a foreign gene producehigher antibody levels to the foreign gene product compared to miceinoculated with a similar recombinant vaccinia virus in which the u geneis intact (Zhou et al., 1990). The u region is present in a defectivenonfunctional form in Copenhagen strain of vaccinia virus (open readingframes B13 and B14 by the terminology reported in Goebel et al.,1990a,b).

Cowpox virus is localized in infected cells in cytoplasmic A typeinclusion bodies (ATI) (Kato et al., 1959). The function of ATI isthought to be the protection of cowpox virus virions duringdissemination from animal to animal (Bergoin et al., 1971). The ATIregion of the cowpox genome encodes a 160 kDa protein which forms thematrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987).Vaccinia virus, though containing a homologous region in its genome,generally does not produce ATI. In WR strain of vaccinia, the ATI regionof the genome is translated as a 94 kDa protein (Patel et al., 1988). InCopenhagen strain of vaccinia virus, most of the DNA sequencescorresponding to the ATI region are deleted, with the remaining 3' endof the region fused with sequences upstream from the ATI region to formopen reading frame (ORF) A26L (Goebel et al., 1990a,b).

A variety of spontaneous (Altenburger et al., 1989; Drillien et al.,1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicaliet al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989;Perkus et al., 1986) deletions have been reported near the left end ofthe vaccinia virus genome. A WR strain of vaccinia virus with a 10 kbspontaneous deletion (Moss et al., 1981; Panicali et al., 1981) wasshown to be attenuated by intracranial inoculation in mice (Buller etal., 1985). This deletion was later shown to include 17 potential ORFs(Kotwal et al., 1988b). Specific genes within the deleted region includethe virokine N1L and a 35 kDa protein (C3L, by the terminology reportedin Goebel et al., 1990a,b). Insertional inactivation of N1L reducesvirulence by intracranial inoculation for both normal and nude mice(Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into themedium of vaccinia virus infected cells. The protein contains homologyto the family of complement control proteins, particularly thecomplement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like thecellular C4bp, the vaccinia 35 kDa protein binds the fourth component ofcomplement and inhibits the classical complement cascade (Kotwal et al.,1990). Thus the vaccinia 35 kDa protein appears to be involved in aidingthe virus in evading host defense mechanisms.

The left end of the vaccinia genome includes two genes which have beenidentified as host range genes, K1L (Gillard et al., 1986) and C7L(Perkus et al., 1990). Deletion of both of these genes reduces theability of vaccinia virus to grow on a variety of human cell lines(Perkus et al., 1990).

Two additional vaccine vector systems involve the use of naturallyhost-restricted poxviruses, avipoxviruses. Both fowlpoxvirus (FPV) andcanarypoxvirus (CPV) have been engineered to express foreign geneproducts. Fowlpox virus (FPV) is the prototypic virus of the Avipoxgenus of the Poxvirus family. The virus causes an economically importantdisease of poultry which has been well controlled since the 1920's bythe use of live attenuated vaccines. Replication of the avipox virusesis limited to avian species (Matthews, 1982b) and there are no reportsin the literature of avipoxvirus causing a productive infection in anynon-avian species including man. This host restriction provides aninherent safety barrier to transmission of the virus to other speciesand makes use of avipoxvirus based vaccine vectors in veterinary andhuman applications an attractive proposition.

FPV has been used advantageously as a vector expressing antigens frompoultry pathogens. The hemagglutinin protein of a virulent avianinfluenza virus was expressed in an FPV recombinant (Taylor et al.,1988a). After inoculation of the recombinant into chickens and turkeys,an immune response was induced which was protective against either ahomologous or a heterologous virulent influenza virus challenge (Tayloret al., 1988a). FPV recombinants expressing the surface glycoproteins ofNewcastle Disease Virus have also been developed (Taylor et al., 1990;Edbauer et al., 1990).

Despite the host-restriction for replication of FPV and CPV to aviansystems, recombinants derived from these viruses were found to expressextrinsic proteins in cells of nonavian origin. Further, suchrecombinant viruses were shown to elicit immunological responsesdirected towards the foreign gene product and where appropriate wereshown to afford protection from challenge against the correspondingpathogen (Tartaglia et al., 1993 a,b; Taylor et al., 1992; 1991b;1988b).

Thus, heretofore, the nucleotide and amino acid sequences for the CHVgB, gC and gD glycoproteins, have not been taught or suggested and,providing these sequences would be of great value. Further, vaccine,antigenic or immunological compositions from the nucleotides for the CHVgB, gC and gD glycoproteins (such as from vector systems containing suchnucleotides) as well as from the glycoproteins themselves (such as fromexpression by the vector systems) have not heretofore been taught orsuggested and, these nucleotides, vector systems, glycoproteins andcompositions would be of great value.

OBJECTS AND SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide nucleotides orisolated nucleic acids coding for CHV gB, gC and gD.

It is a further object of the invention to provide vectors containingnucleotides or isolated nucleic acids coding for CHV gB, gC and/or gD.

It is another object of the invention to provide CHV gB, gC and/or gDglycoproteins, especially from expression of nucleotides or isolatednucleic acids therefor in a vector system.

It is an additional object of the invention to provide antigenic,vaccine or immunological compositions from the CHV gB, gC and/or gDnucleotides or isolated nucleic acids or a vector containing them or,from the glycoproteins themselves, such as by way of expression by thevector.

It is yet another object of this invention to provide modifiedrecombinant viruses, which viruses have enhanced safety, and to providea method of making such recombinant viruses.

It is an additional object of this invention to provide a recombinantpoxvirus antigenic, vaccine or immunological composition having anincreased level of safety compared to known recombinant poxvirusantigenic, vaccine or immunological compositions.

It is a further object of this invention to provide a modified vectorfor expressing a gene product in a host, wherein the vector is modifiedso that it has attenuated virulence in the host.

It is another object of this invention to provide a method forexpressing a gene product, such as CHV gB, gC and/or gD, in a cellcultured in vitro using a modified recombinant virus or modified vectorhaving an increased level of safety.

These and other objects and advantages of the present invention willbecome more readily apparent after consideration of the following.

The present invention involves the elucidation of the CHV gB, gC and gDnucleotides, glycoproteins therefrom and, antigenic, vaccine orimmunological compositions employing the nucleotide sequences and theglycoproteins.

Accordingly, the present invention provides a nucleotide or isolatednucleic acid coding for canine herpesvirus gB glycoprotein.

The present invention provides a nucleotide or isolated nucleic acidcoding for canine herpesvirus gC glycoprotein.

The present invention provides a nucleotide or isolated nucleic acidcoding for canine herpesvirus gD glycoprotein.

The nucleotides are preferably DNA. The nucleotides or isolated nucleicacids preferably have the DNA sequences as set forth in FIGS. 1, 4 and7.

The present invention also provides canine herpesvirus glycoprotein gB.

The present invention provides canine herpesvirus glycoprotein gC.

The present invention provides canine herpesvirus glycoprotein gD.

The present invention further provides a vector containing thenucleotide or isolated nucleic acid for canine herpesvirus gB, gC and/orgD. Preferably the vector is a recombinant poxvirus such as arecombinant vaccinia or avipox virus, more preferably the vaccinia oravipox virus is attenuated such as NYVAC, ALVAC or TROVAC.

Thus, in one preferred aspect, the present invention relates to amodified recombinant virus having inactivated virus-encoded geneticfunctions so that the recombinant virus has attenuated virulence andenhanced safety. The functions can be non-essential, or associated withvirulence. The virus is advantageously a poxvirus, particularly avaccinia virus or an avipox virus, such as fowlpox virus and canarypoxvirus. The modified recombinant virus can include, within anon-essential region of the virus genome, a heterologous DNA sequencewhich encodes a CHV antigenic protein, e.g., CHV gC, gB, and gD or anycombination thereof.

In a still further preferred aspect, the present invention relates to amodified recombinant virus having nonessential virus-encoded geneticfunctions inactivated therein so that the virus has attenuatedvirulence, and wherein the modified recombinant virus further containsDNA from a heterologous source in a nonessential region of the virusgenome. The DNA can code for a CHV gB, gC and gD, or any combinationthereof. In particular, the genetic functions are inactivated bydeleting an open reading frame encoding a virulence factor or byutilizing naturally host restricted viruses. The virus used according tothe present invention is advantageously a poxvirus, particularly avaccinia virus or an avipox virus, such as fowlpox virus and canarypoxvirus. Advantageously, the open reading frame is selected from the groupconsisting of J2R, B13R+B14R, A26L, A56R, C7L-K1L, and I4L (by theterminology reported in Goebel et al., 1990a,b); and, the combinationthereof. In this respect, the open reading frame comprises a thymidinekinase gene, a hemorrhagic region, an A type inclusion body region, ahemagglutinin gene, a host range gene region or a large subunit,ribonucleotide reductase; or, the combination thereof. The modifiedCopenhagen strain of vaccinia virus is identified as NYVAC (Tartaglia etal., 1992).

The present invention still further provides an antigenic, vaccine orimmunological composition for inducing an antigenic or immunologicalresponse in a host, such as a canine, comprising a suitable vectorcontaining the nucleotide(s) or isolated nucleic acid(s) for canineherpesvirus gB, gC and/or gD and a suitable carrier; or, canineherpesvirus gB, gC and/or gD glycoprotein(s), such as from expressionthereof in a vector containing the nucleotide(s) of the invention, and asuitable carrier.

The present invention yet further provides methods employing theinventive nucleotide(s) or isolated nucleic acid(s), glycoprotein(s),composition(s).

Thus, the invention provides a method for preparing canine herpesvirusgB, gC and/or gD comprising inserting the nucleotide(s) or isolatednucleic acid(s) therefor into a suitable vector, cultivating the vector,and, collecting the glycoprotein from the vector. The vector can be apoxvirus, such as vaccinia or avipox virus, a phage such as lambda, orE. coli or any other suitable virus or bacterial vector. The cultivatingcan be infecting cells susceptible to viral infection by the virusvector or, growing colonies of the bacterial vector system, such as byplate or broth methods. And, collecting can be by separating theglycoprotein(s) from the viral-infected cells or from the bacterialcells.

Thus, in a preferred aspect, the present invention relates to a methodfor expressing a gene product in a cell cultured in vitro by introducinginto the cell a modified recombinant virus having attenuated virulenceand enhanced safety. The modified recombinant virus can include, withina non-essential region of the virus genome, a heterologous DNA sequencewhich encodes an antigenic protein, e.g., CHV gB, gC and gD, or anycombination thereof.

Likewise, the invention provides a method for inoculating or forstimulating an antigenic or immunological response in a host such as acanine against canine herpesvirus comprising administering the inventiveantigenic, vaccine or immunological composition to the host, e.g.,canine. Additionally, the invention includes an antibody elicited by theexpression of the inventive nucleotide(s). The antibody can be generatedinto a monoclonal antibody by known techniques and, the antibody or themonoclonal antibody can be employed in a binding diagnostic assay, testor kit to determine the presence or absence of CHV gB, gC and/or gD in asample such as sera and therefore the presence or absence of CHV or, ofan antibody or immune response to CHV or to glycoproteins thereof.

These and other embodiments within the present invention are describedor are obvious from the following detailed description.

BRIEF DESCRIPTION OF DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows the nucleotide sequence and predicted amino acid sequenceof the CHV gB homologue (SEQ ID NOS:1, 2);

FIG. 2 shows the hydropathicity analysis of the CHV gB homologue;

FIGS. 3A and 3B show the amino acid homology of 8 gB homologues (SEQ IDNOS:3-10);

FIG. 4 shows the nucleotide sequence and predicted amino acid sequenceof the CHV gC homologue and ORF2 (SEQ ID NOS:11-13);

FIG. 5 shows the hydropathicity analysis of the CHV gC homologue;

FIG. 6 shows the amino acid homology of 4 gC homologues (SEQ IDNOS:14-17);

FIG. 7 shows the nucleotide sequence and predicted amino acid sequenceof the CHV gD homologue; SEQ ID NOS:18-20);

FIG. 8 shows the hydropathicity analysis of the CHV gD homologue;

FIG. 9 shows amino acid homology of 4 gD homologues (SEQ ID NOS:16-20);

FIG. 10 schematically shows a method for the construction of plasmidpSD460 for deletion of thymidine kinase gene and generation ofrecombinant vaccinia virus vP410;

FIG. 11 schematically shows a method for the construction of plasmidpSD486 for deletion of hemorrhagic region and generation of recombinantvaccinia virus vP553;

FIG. 12 schematically shows a method for the construction of plasmidpMP494Δ for deletion of ATI region and generation of recombinantvaccinia virus vP618;

FIG. 13 schematically shows a method for the construction of plasmidpSD467 for deletion of hemagglutinin gene and generation of recombinantvaccinia virus vP723;

FIG. 14 schematically shows a method for the construction of plasmidpMPCK1Δ for deletion of gene cluster [C7L-K1L] and generation ofrecombinant vaccinia virus vP804;

FIG. 15 schematically shows a method for the construction of plasmidpSD548 for deletion of large subunit, ribonucleotide reductase andgeneration of recombinant vaccinia virus vP866 (NYVAC);

FIG. 16 schematically shows a method for the construction of plasmidpRW842 for insertion of rabies glycoprotein G gene into the TK deletionlocus and generation of recombinant vaccinia virus vP879;

FIG. 17 shows the DNA sequence (SEQ ID NO:62) of a canarypox PvuIIfragment containing the C5 ORF.

FIGS. 18A and 18B schematically show a method for the construction ofrecombinant canarypox virus vCP65 (ALVAC-RG);

FIG. 19 shows schematically the ORFs deleted to generate NYVAC;

FIG. 20 shows the nucleotide sequence (SEQ ID NO:72) of a fragment ofTROVAC DNA containing an F8 ORF;

FIG. 21 shows the DNA sequence (SEQ ID NO:75) of a 2356 base pairfragment of TROVAC DNA containing the F7 ORF;

FIGS. 22A to 22D show graphs of rabies neutralizing antibody titers(RFFIT, IU/ml), booster effect of HDC and vCP65 (10⁵.5 TCID50) involunteers previously immunized with either the same or the alternatevaccine (vaccines given at days 0, 28 and 180, antibody titers measuredat days 0, 7, 28, 35, 56, 173, 187 and 208);

FIG. 23 shows the nucleotide sequence of the I3L-promoted CHV gB genecontained in pCHV37 and vCP320;

FIG. 24 shows the nucleotide sequence of the ALVAC C6 flanking arms;

FIG. 25 shows the immunoprecipitation analysis of vCP320-infected cells(Lysates from ³⁵ S-labeled mock-infected cells (lane A), ALVAC-infectedcells (lane B), vCP320-infected cells (lane C) and CHV-infected cells(lane D) were immunoprecipitated with a CHV gB-specific monoclonalantibody, 1125B2 (obtained from Rhone Merieux, Lyon, France), andresolved on an SDS-polyacrylamide gel. Molecular weight standards areresolved in lane E);

FIG. 26 shows the nucleotide sequence of the H6-promoted CHV gC genecontained in pCHV40 and vCP322;

FIG. 27 shows the immunoprecipitation analysis of vCP322-infected cells(Lysates from ³⁵ S-labeled mock-infected cells (lane A), ALVAC-infectedcells (lane B), vCP322-infected cells (lane C) and CHV-infected cells(lane D) were immunoprecipitated with a CHV gC-specific monoclonalantibody, 2011A9 (obtained from Rhone Merieux, Lyon, France), andresolved on an SDS-polyacrylamide gel. Molecular weight standards areresolved in lane E);

FIG. 28 shows the nucleotide sequence of the H6-promoted CHV gD genecontained in pCHV26 and vCP294; and

FIG. 29 shows the immunoprecipitation analysis of vCP294-infected cells(Lysates from ³⁵ S-labeled mock-infected cells (lane A), ALVAC-infectedcells (lane B), vCP294-infected cells (lane C) and CHV-infected cells(lane D) were immunoprecipitated with a CHV gD-specific monoclonalantibody, 208D11 (obtained from Rhone Merieux, Lyon, France), andresolved on an SDS-polyacrylamide gel. Molecular weight standards areresolved in lane E).

DETAILED DESCRIPTION

This invention provides nucleotides coding for the CHV gB, gC and gDgenes. These genes encode polypeptides of 879, 459 and 345 amino acids,respectively. Comparison of the predicted amino acid sequence of theseglycoproteins with the gB, gC and gD amino acid sequences of otherherpesviruses indicates that CHV is an alpha-herpesvirus; a conclusionthat is consistent with the previous classification of this virusaccording to biological properties. This analysis also revealed that thehomology among gB homologues is greater than the homology among gC or gDhomologues, suggesting that the structural and functional constraints ongB may be greater than those on gC or gD.

Alignment of homologous gB, gC and gD polypeptides revealed that thevast majority of cysteine residues are perfectly conserved. Theseresults suggest that these cysteine residues, due to their ability toform disulfide bonds, are important in maintaining the structural andfunctional integrity of the gB, gC and gD glycoproteins. In fact, inHSV1 gD, it has been shown that cysteine 1 forms a disulfide bond withcysteine 5, cysteine 2 forms a disulfide bond with cysteine 6 andcysteine 3 forms a disulfide bond with cysteine 4 (Long et al., 1992).Furthermore, it has been shown that a mutation of any of these residueshas a profound effect on the conformation, processing and function ofthe resulting glycoprotein (Wilcox et al., 1988; Long et al., 1990).Therefore, the conservation of cysteine residues in the glycoproteins ofthe invention may also have structural significance.

The high degree of homology among the gC, gD and, in particular, gBhomologues also suggests that these glycoproteins have common functions.In fact, it has been shown that the BHV1 gB homologue can rescue a gB⁻PRV virus, indicating that these 2 glycoproteins are functionallyequivalent (Kopp & Mettenleiter, 1992).

Alignment of the gB, gC and gD amino acid sequences also revealed thatpotential N-linked glycosylation sites are somewhat conserved. N-linkedglycosylation is thought to play a role in a variety of functions, suchas maintenance of protein conformation and protection againstproteolytic degradation. The biological significance of N-linkedcarbohydrates on herpesvirus glycoproteins, however, is not completelyunderstood. For example, tunicamycin treatment of HSV1 infected cellshas been shown to inhibit the production of infectious virions (Pizer etal., 1980). In addition, endoglycosidase treatment of HSV1 virions hasbeen shown to decrease infectivity (Kuhn et al., 1988). On the otherhand, N-linked glycosylation of HSV1 gD does not appear to be absolutelyessential, since mutagenesis of the glycosylation sites on thisglycoprotein does not affect infectivity (Sodora et al., 1991).Therefore, although the glycosylation sites on the gB, gC and gDglycoproteins are relatively well conserved, proper glycosylation ofeach of these polypeptides may not be absolutely essential.

The G+C content of herpesviruses varies from 33%-75% (Roizman, 1982). Ithas been suggested that this extensive variability is due to anonselective mutational bias based on the presence (or absence) ofvirally encoded or induced enzymes involved in nucleotide metabolism(Honess, 1984). For example, VZV and herpesvirus saimiri (HVS) both haverelatively low G+C contents (46% and 46%, respectively) and both encodean enzyme, thymidylate synthetase, which is involved in TTP synthesis(Davison & Scott, 1986; Honess et al., 1986). HSV1, HCMV and EBV, on theother hand, have relatively high G+C contents (68%, 57% and 60%,respectively) and do not appear to encode a thymidylate synthetase(Honess et al., 1986). CHV has been determined by DNA density analysisto have the lowest G+C content of any herpesvirus, 33% (Plummer et al.,1969; Roizman, 1982); a value which is consistent with the relativelylow G+C content of the nucleotides of the invention (29%). Withoutwishing it to be bound by the theory that CHV does not encode an enzymeinvolved in nucleotide metabolism, from the present invention the ORFlocated immediately downstream from the CHV gC gene is not homologous toVZV thymidylate synthetase. Therefore, if CHV contains a thymidylatesynthetase gene, it is not found at the same genomic location as VZV.

Newborn pups exposed to CHV usually die without forming CHV-specificneutralizing antibodies. Also, the maternal antibodies or treatment withimmune serum from seropositive dogs can protect pups from a fatal CHVinfection (Carmichael, 1970). Therefore, serum neutralizing antibodiescan protect pups against a fatal CHV infection. Likewise, serumneutralizing antibodies can protect adult dogs from the self-limitingsubclinical, upper respiratory tract infection.

Three CHV glycoproteins, gp145/112, gp80 and gp47, are known to elicitCHV neutralizing antibodies (Xuan et al., 1991). The genes encodingthese glycoproteins have not been identified. Without wishing to bebound by any one theory, it is possible, however, that these antigensare encoded by the gB, gC and gD genes of this invention. Since severalreports have indicated that an immune response against gB, gC and/or gDcan provide protection of target species animals against a herpesviruschallenge (Babiuk et al., 1987; Nazarian et al., 1992; Riviere et al.,1992; Brockmeier et al., 1993), the CHV gB, gC and gD genes of thisinvention provide efficacious CHV glycoproteins, immunological orvaccine compositions and methods of using the same.

In particular, the nucleotides of this invention can be inserted intoany suitable vector system for expression. For instance, thenucleotide(s) can be inserted into any suitable bacterial vector systemsuch as the E. coli system, employing known methods (see, e.g., Robbins,EPA 0162738A1; Panicali, EPA 0261940A2).

The nucleotide(s) can be inserted into any suitable phage or viralvector system such as lambda, poxvirus, herpesvirus (see Roizman, U.S.Pat. No. 4,769,331, incorporated herein by reference), baculovirus,polio virus (see Kitson et al., J. Virol. 65, 3068-3075, 1991,incorporated herein by reference), and adenovirus (see Grunhaus et al.,1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p.237-52, 1993; Ballay et al., EMBO Journal, vol. 4, p. 3861-65; Graham,Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen. Virol. 70,429-434, each of which is incorporated herein by reference) systemsemploying known methods.

The preferred vector system is a poxvirus vector system, especially anavipox vaccinia virus system wherein recombination is as in U.S. Pat.Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587 and 5,179,993. However,an attenuated poxvirus system is even more preferred.

To develop a new vaccinia vaccine strain, NYVAC (vP866), the Copenhagenvaccine strain of vaccinia virus was modified by the deletion of sixnonessential regions of the genome encoding known or potential virulencefactors. The sequential deletions are detailed below. All designationsof vaccinia restriction fragments, open reading frames and nucleotidepositions are based on the terminology reported in Goebel et al.,1990a,b.

The deletion loci were also engineered as recipient loci for theinsertion of foreign genes.

The regions deleted in NYVAC are listed below. Also listed are theabbreviations and open reading frame designations for the deletedregions (Goebel et al., 1990a,b) and the designation of the vacciniarecombinant (vP) containing all deletions through the deletionspecified:

(1) thymidine kinase gene (TK; J2R) vP410;

(2) hemorrhagic region (u; B13R+B14R) vP553;

(3) A type inclusion body region (ATI; A26L) vP618;

(4) hemagglutinin gene (HA; A56R) vP723;

(5) host range gene region (C7L-K1L) vP804; and

(6) large subunit, ribonucleotide reductase (I4L) vP866 (NYVAC).

NYVAC is a genetically engineered vaccinia virus strain that wasgenerated by the specific deletion of eighteen open reading framesencoding gene products associated with virulence and host range. NYVACis highly attenuated by a number of criteria including i) decreasedvirulence after intracerebral inoculation in newborn mice, ii) inocuityin genetically (nu⁺ /nu⁺) or chemically (cyclophosphamide)immunocompromised mice, iii) failure to cause disseminated infection inimmunocompromised mice, iv) lack of significant induration andulceration on rabbit skin, v) rapid clearance from the site ofinoculation, and vi) greatly reduced replication competency on a numberof tissue culture cell lines including those of human origin.Nevertheless, NYVAC based vectors induce excellent responses toextrinsic immunogens and provided protective immunity.

TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolatederived from the FP-1 vaccine strain of fowlpoxvirus which is licensedfor vaccination of 1 day old chicks. ALVAC is an attenuated canarypoxvirus-based vector that was a plaque-cloned derivative of the licensedcanarypox vaccine, Kanapox (Tartaglia et al., 1992). ALVAC has somegeneral properties which are the same as some general properties ofKanapox. ALVAC-based recombinant viruses expressing extrinsic immunogenshave also been demonstrated efficacious as vaccine vectors (Tartaglia etal., 1993 a,b). This avipox vector is restricted to avian species forproductive replication. On human cell cultures, canarypox virusreplication is aborted early in the viral replication cycle prior toviral DNA synthesis. Nevertheless, when engineered to express extrinsicimmunogens, authentic expression and processing is observed in vitro inmammalian cells and inoculation into numerous mammalian species inducesantibody and cellular immune responses to the extrinsic immunogen andprovides protection against challenge with the cognate pathogen (Tayloret al., 1992; Taylor et al., 1991). Recent Phase I clinical trials inboth Europe and the United States of a canarypox/rabies glycoproteinrecombinant (ALVAC-RG) demonstrated that the experimental vaccine waswell tolerated and induced protective levels of rabiesvirus neutralizingantibody titers (Cadoz et al., 1992; Fries et al., 1992). Additionally,peripheral blood mononuclear cells (PBMCS) derived from the ALVAC-RGvaccinates demonstrated significant levels of lymphocyte proliferationwhen stimulated with purified rabies virus (Fries et al., 1992).

NYVAC, ALVAC and TROVAC have also been recognized as unique among allpoxviruses in that the National Institutes of Health ("NIH") (U.S.Public Health Service), Recombinant DNA Advisory Committee, which issuesguidelines for the physical containment of genetic material such asviruses and vectors, i.e., guidelines for safety procedures for the useof such viruses and vectors which are based upon the pathogenicity ofthe particular virus or vector, granted a reduction in physicalcontainment level: from BSL2 to BSL1. No other poxvirus has a BSL1physical containment level. Even the Copenhagen strain of vacciniavirus--the common smallpox vaccine--has a higher physical containmentlevel; namely, BSL2. Accordingly, the art has recognized that NYVAC,ALVAC and TROVAC have a lower pathogenicity than any other poxvirus.

Clearly based on the attenuation profiles of the NYVAC, ALVAC, andTROVAC vectors and their demonstrated ability to elicit both humoral andcellular immunological responses to extrinsic immunogens (Tartaglia etal., 1993a,b; Taylor et al., 1992; Konishi et al., 1992) suchrecombinant viruses offer a distinct advantage over previously describedvaccinia-based recombinant viruses.

After growing the bacteria or infecting cells with the recombinantvirus, the glycoprotein(s) are collected by known techniques such aschromatography (see Robbins, EPA 0162738A1; Panicali, EPA 0261940A2).

The collected glycoprotein(s) can then be employed in a vaccine,antigenic or immunological composition which also contains a suitablecarrier. Accordingly, the inventive nucleotides are quite useful.

Alternatively, the viral vector system, especially the preferredpoxvirus vector system, can be employed in a vaccine, antigenic orimmunological composition which also contains a suitable carrier. TheCHV recombinant poxvirus in the composition expresses the CHVglycoprotein in vivo after administration or inoculation.

The antigenic, immunological or vaccine composition of the invention(either containing glycoprotein(s) expressed from a vector systemcontaining the inventive nucleotide(s) or containing a suitable vectorsystem such as the CHV recombinant poxvirus) is administered to pups inthe same fashion as maternal antibodies or immune serum fromseropositive dogs (Carmichael, 1970). Seronegative dogs are administeredthe composition in the same fashion as other antigenic, vaccine orimmunological compositions are administered. One skilled in theveterinary arts can determine dosage from this disclosure without undueexperimentation, taking into consideration such factors as the age,weight, breed, sex and general health of the particular dog or pup.

Additionally, the inventive recombinant poxvirus and the expressionproducts therefrom stimulate an immune or antibody response in animals.From those antibodies, by techniques well-known in the art, monoclonalantibodies can be prepared and, those monoclonal antibodies or theantigens expressed from the inventive nucleotides, can be employed inwell known antibody binding assays, diagnostic kits or tests todetermine the presence or absence of particular CHV gB, gC and/or gDantigen(s) or antibodies thereto and therefrom the presence or absenceof the virus or, to determine whether an immune response to the virus orantigen(s) has simply been stimulated.

Monoclonal antibodies are immunoglobulins produced by hybridoma cells. Amonoclonal antibody reacts with a single antigenic determinant andprovides greater specificity than a conventional, serum-derivedantibody. Furthermore, screening a large number of monoclonal antibodiesmakes it possible to select an individual antibody with desiredspecificity, avidity and isotype. Hybridoma cell lines provide aconstant, inexpensive source of chemically identical antibodies andpreparations of such antibodies can be easily standardized. Methods forproducing monoclonal antibodies are well known to those of ordinaryskill in the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265,issued Apr. 1, 1989, incorporated herein by reference.

Uses of monoclonal antibodies are known. One such use is in diagnosticmethods, e.g., David, G. and Greene, H., U.S. Pat. No. 4,376,110, issuedMar. 8, 1983, incorporated herein by reference.

Monoclonal antibodies have also been used to recover materials byimmunoadsorption chromatography, e.g. Milstein, C., 1980, ScientificAmerican 243:66, 70, incorporated herein by reference.

Additionally, the inventive nucleotides can be used as probes toascertain the presence of CHV DNA in samples, as well as in thegeneration of PCR primers for replicating or cloning CHV DNA. Methodsfor using DNA as probes or for preparing PCR primers are known in theart.

Thus, the inventive nucleotides and expression products of the inventivenucleotides (and therefore the nucleotides) are quite useful.

The following non-limiting Examples are given by way of illustrationonly and are not to be considered a limitation of this invention.

EXAMPLES Methods and Materials

Preparation of genomic CHV DNA. CHV (obtained from L. Carmichael,Cornell University) was propagated on Madin-Darby canine kidney (MDCK)cells (ATCC CCL34). Viral DNA was isolated by standard methodology(Tartaglia et al., 1990).

DNA Hybridization.

CHV genomic DNA was digested with restriction enzymes, run on agarosegels and transferred to Gene-Screen membranes (New England Nuclear)under conditions recommended by the manufacturers. Hybridizations wereperformed at 44° C., 53° C. or 59° C. in 1M NaCl, 1% SDS and 10% dextransulfate. The hybridization probe included a 1800 bp BamHI-XbaI fragment,containing an internal segment of the feline herpesvirus (FHV) gB gene,a 950 bp BamHI-EcoRI fragment, containing the 3'-end of the FHV gC geneand a 970 bp BamHI-HindIII fragment, containing the 3'-end of the FHV gDgene (Audonnet, unpublished results).

Cloning and DNA Sequencing.

CHV genomic fragments were subcloned into pBluescriptSK (Stratagene).Plasmid DNA was prepared and manipulated using standard techniques.Nucleotide sequencing was performed on double-stranded plasmidtemplates, using the modified T7 enzyme, Sequenase (U.S. BiochemicalCorporation), and standard protocols recommended by the manufacturer.M13 forward and reverse primers were used to obtain initial sequence,and custom primers, prepared with a Biosearch 8700 or an AppliedBiosystems 380B oligonucleotide synthesizer, were used for subsequentreactions.

DNA and Amino Acid Sequence Analyses.

DNA and amino acid sequence analyses were performed with PC/GENE(IntelliGenetics, Incorporated), ALIGN Plus (Scientific and EducationalSoftware) and IBI-Pustell (International Biotechnologies, Incorporated)software packages. Homology searches were conducted on the SWISS-PROT(Release 20 or 23) (IntelliGenetics, Incorporated) database, using theFASTA program (Pearson & Lipman, 1988).

DNA Cloning and Synthesis.

Plasmids were constructed, screened and grown by standard procedures(Maniatis et al., 1982; Perkus et al., 1985; Piccini et al., 1987).Restriction endonucleases were obtained from Bethesda ResearchLaboratories, Gaithersburg, Md., New England Biolabs, Beverly, Mass.;and Boehringer Mannheim Biochemicals, Indianapolis, Ind. Klenow fragmentof E. coli polymerase was obtained from Boehringer MannheimBiochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtainedfrom New England Biolabs. The reagents were used as specified by thevarious suppliers.

Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 orApplied Biosystems 380B DNA synthesizer as previously described (Perkuset al., 1989). DNA sequencing was performed by the dideoxy-chaintermination method (Sanger et al., 1977) using Sequenase (Tabor et al.,1987) as previously described (Guo et al., 1989). DNA amplification bypolymerase chain reaction (PCR) for sequence verification (Engelke etal., 1988) was performed using custom synthesized oligonucleotideprimers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus,Norwalk, Conn.) in an automated Perkin Elmer Cetus DNA Thermal Cycler.Excess DNA sequences were deleted from plasmids by restrictionendonuclease digestion followed by limited digestion by BAL-31exonuclease and mutagenesis (Mandecki, 1986) using syntheticoligonucleotides.

Cells, Virus, and Transfection.

The origins and conditions of cultivation of the Copenhagen strain ofvaccinia virus has been previously described (Guo et al., 1989).Generation of recombinant virus by recombination, in situ hybridizationof nitrocellulose filters and screening for B-galactosidase activity areas previously described (Piccini et al., 1987).

The origins and conditions of cultivation of the Copenhagen strain ofvaccinia virus and NYVAC has been previously described (Guo et al.,1989; Tartaglia et al., 1992). Generation of recombinant virus byrecombination, in situ hybridization of nitrocellulose filters andscreening for B-galactosidase activity are as previously described(Panicali et al., 1982; Perkus et al., 1989).

The parental canarypox virus (Rentschler strain) is a vaccinal strainfor canaries. The vaccine strain was obtained from a wild type isolateand attenuated through more than 200 serial passages on chick embryofibroblasts. A master viral seed was subjected to four successive plaquepurifications under agar and one plaque clone was amplified through fiveadditional passages after which the stock virus was used as the parentalvirus in in vitro recombination tests. The plaque purified canarypoxisolate is designated ALVAC.

The strain of fowlpox virus (FPV) designated FP-1 has been describedpreviously (Taylor et al., 1988a). It is an attenuated vaccine strainuseful in vaccination of day old chickens. The parental virus strainDuvette was obtained in France as a fowlpox scale from a chicken. Thevirus was attenuated by approximately 50 serial passages in chickenembryonated eggs followed by 25 passages on chicken embryo fibroblastcells. The virus was subjected to four successive plaque purifications.One plaque isolate was further amplified in primary CEF cells and astock virus, designated as TROVAC, established.

NYVAC, ALVAC and TROVAC viral vectors and their derivatives werepropagated as described previously (Piccini et al., 1987; Taylor et al.,1988a,b). Vero cells and chick embryo fibroblasts (CEF) were propagatedas described previously (Taylor et al., 1988a,b).

Example 1 Identification and Sequencing of the CHV gB Gene

Hybridization of CHV genomic DNA at relatively low stringency with aradiolabelled probe containing the feline herpesvirus (FHV) gB, gC andgD genes (Audonnet, unpublished results) identified one complimentarysequence. A 6 kb XbaI fragment containing this sequence was cloned andthe nucleotide sequence of the hybridizing region was determined. Thesequence of the nucleotide coding the CHV gB gene is shown in FIG. 1together with the predicted amino acid expression (gB glycoprotein)therefrom. The putative transmembrane regions and potential TATA, CAATand polyadenylation signal sequences are underlined. Nucleotides andpredicted amino acid residues are numbered to the right of the sequence.

An open reading frame (ORF) starting at position 201 and ending atposition 2840 was identified. The translation product (predicted) ofthis ORF is 879 amino acids long. Comparison of this amino acid sequencewith the SWISS-PROT (Release 20) database revealed significant homologywith the gB glycoprotein of numerous herpesviruses. Additional analysesrevealed that the CHV gene product (predicted) was more homologous tothe gB glycoprotein of alpha-herpesviruses, such as herpes simplex virustype 1 (HSV1), than beta- or gamma-herpesviruses, such as humancytomegalovirus (HCMV) or Epstein-Barr virus (EBV). These analyses andthe results thereof are shown in Table 1 below. These results indicatethat CHV should be classified as an alpha-herpesvirus; a conclusion thatis consistent with the previous classification of this virus accordingto biological properties (Carmichael et al., 1965; Roizman, 1982).

                                      TABLE 1                                     __________________________________________________________________________    HOMOLOGY BETWEEN THE PREDICTED AMINO ACID                                     SEQUENCES OF 10 HERPESVIRUS gB GLYCOPROTEINS                                  FHV     EHV1                                                                              PRV BHV1                                                                              VZV MDV HSV1                                                                              HCMV                                                                              EBV                                       __________________________________________________________________________    CHV 78  61  61  59  55  52  50  29  27                                        FHV     57  59  58  51  48  48  27  27                                        EHV1        52  52  47  45  44  27  26                                        PRV             63  52  48  50  29  29                                        BHV1                52  46  48  28  28                                        VZV                     49  48  30  28                                        MDV                         48  30  29                                        HSV1                            28  29                                        HCMV                                32                                        __________________________________________________________________________

Values in Table 1 were obtained using the ALIGN Plus program and areexpressed as percent homology. The entire gB amino acid sequence wasused. The alignment parameters were: mismatch penalty=2, open gappenalty=4, extended gap penalty=1. References: FHV (Maeda et al., 1992),EHV1 (Whalley et al., 1989), PRV (Robbins et al., 1987), BHV1 (Whitbecket al., 1988), VZV (Keller et al., 1986), MDV (Ross et al., 1989), HSV1(Bzik et al., 1984), HCMV (Kouzarides et al., 1987) and EBV (Pellett etal., 1985).

Example 2 Analysis of the CHV gB Nucleotide Sequence

The 5'- and 3'-noncoding regions of the CHV gB gene contain numerous RNApolymerase II regulatory sequence motifs, such as TATA box, CAAT box andpolyadenylation signal sequences (Corden et al., 1980; Proudfoot &Brownlee, 1976) (FIG. 1). Potential TATA box sequences are found atpositions 34, 36, 119 and 148, approximately 165 bp, 160 bp, 80 bp and50 bp upstream from the CHV gB initiation codon. Potential CAAT boxsequences (ATTG) are found at positions 89, 97 and 165, approximately110 bp, 100 bp and 35 bp upstream from the gB initiation codon.Potential polyadenylation signal sequences (AATAAA) are found atpositions 2839 and 2961, approximately 0 bp and 120 bp downstream fromthe CHV gB termination codon.

The nucleotide sequence surrounding the initiation codon has been shownto affect the efficiency of translation initiation (Kozak, 1986). Inparticular, the sequence, [A/G]NNATGG, has been found to be mostefficient. Therefore, relative to Kozak's rules, the nucleotide sequencesurrounding the CHV gB initiation codon (AGTATGT) is favorable atposition -3, but not at position +4 (FIG. 1). The fact that the CHV gBgene does not follow Kozak's rules is not unusual. The FHV (Maeda etal., 1992), PRV (Robbins et al., 1987), varicella-zoster virus (VZV)(Keller et al., 1986), MDV (Ross et al., 1989) and HSV1 (Bzik et al.,1984) gB genes also contain a pyrimidine at position +4.

Example 3 Analysis of the Predicted CHV gB Amino Acid Sequence

The deduced amino acid sequence of the CHV gB homologue is presented inFIG. 1. Hydropathicity analysis of this amino acid sequence is shown inFIG. 2. The profile was obtained with the PC/GENE SOAP program, usingthe method of Kyte & Doolittle (1982) and an interval of 13 amino acids.The vertical axis represents relative hydropathicity, where positivevalues are hydrophobic and negative values are hydrophilic. Thehorizontal axis represents the amino acid number of the CHV gBhomologue.

Hydropathicity analysis of this amino acid sequence revealed thepresence of 2 prominent hydrophobic peaks. The first peak, located atthe N-terminus, without wishing to be bound by any one theory,represents a potential signal sequence. N-terminal signal sequencesinitiate transport across the endoplasmic reticulum membrane and can becritical for the proper post-translational modification and targeting ofglycoproteins (Blobel, 1980). Signal sequences vary in length from about15-30 residues and usually consist of a basic N-terminal region, acentral hydrophobic region and a short, relatively polar C-terminalregion. In addition, the cleavage site usually conforms to the -3,-1rule, where the residue at position -1 is small (Ala, Ser, Gly, Cys, Thror Gln) and the residue at position -3 is not aromatic (Phe, His, Tyr orTrp), charged (Asp, Glu, Lys or Arg) or large and polar (Asn or Gln),and residues -3 through +1 are not Pro (von Heijne, 1986). Althoughanalysis with PSIGNAL, a PC/GENE program designed to detect eukaryoticsignal sequences, does not identify the N-terminal end of CHV gB as apotential signal sequence, this region does have elements consistentwith typical signal sequences; namely a hydrophobic core (residues 2-17)and a relatively polar C-terminal region (FIG. 1). The fact that PSIGNALdoes not detect a signal sequence in the N-terminal region of CHV gB isnot unique. This algorithm also does not detect a signal sequence in theN-terminal region of the VZV gB homologue.

The second, very broad, hydrophobic peak(s) (FIG. 2), with predictedmembrane-spanning segments between amino acid residues 725 and 741 and746-750 and 766-772 (using the method of Klein et al. (1985)), withoutwishing to be bound by any one theory, functions as a membrane anchorregion. It has been hypothesized that the transmembrane domain of HSV1gB, as well as other gB homologues, transverses the membrane 3 times(Pellett et al., .1985). Hydropathicity analysis of CHV gB reveals thepresence of at least 2 distinct hydrophobic peaks. Therefore, CHV gB andHSV1 gB have similar transmembrane structures.

Alignment of the CHV gB amino acid sequence with similar sequences fromother herpesviruses revealed extensive homology throughout the entiresequence, with the exception of the N-terminus, a region surrounding theputative cleavage site (see below) and a region near the C-terminus.FIGS. 3A and 3B show the amino acid homology of 8 gB homologues. Theamino acid sequences of the CHV, FHV, EHV1, PRV, HSV1, VZV, HCMV and EBVgB homologues (for references from which the sequences were obtained,see text below Table 1) were aligned using the PC/GENE CLUSTAL program.Gaps, indicated by dashes, were introduced to maximize homology. Alignedresidues which are identical in all 8 sequences are indicated by anasterisk (*). Aligned residues which are identical in the majority ofsequences are indicated by a period (.). Conserved cysteine residues areboxed. Potential N-linked glycosylation sites are shaded. Putativeproteolytic cleavage sites are underlined.

This alignment also revealed that the vast majority of cysteine residuesare perfectly conserved. For example, CHV gB contains 11 cysteineresidues, 10 of which are perfectly conserved in all alpha-, beta- andgamma-herpesviruses. In fact, the only cysteine residue in CHV gB thatis not conserved is found near the N-terminus and may be located in theputative signal sequence. These results show that the gB glycoproteinshave relatively similar tertiary structures.

Alignment of the gB amino acid sequences also revealed that thepotential N-linked glycosylation sites are relatively well conserved(FIGS. 3A and 3B). N-linked oligosaccharides can be added to Asnresidues that have the sequence Asn-X-Ser or Asn-X-Thr, where X is notPro (Bause, 1983). CHV gB contains 13 potential N-linked glycosylationsites. Three of these sites, however, are situated in the putativecytoplasmic domain and, therefore, may not be glycosylated. The locationof the potential N-linked glycosylation sites is relatively wellconserved in the majority of gB glycoproteins (FIGS. 3A and 3B).

The gB glycoprotein of most herpesviruses is cleaved internally duringmaturation, with the subsequent peptides being held together bydisulfide bonds. The VZV gB homologue (gpII), for example, is cleavedbetween Arg and Ser residues, resulting in 2 glycoproteins ofapproximately 60 kd (Keller et al., 1986). The gB glycoproteins of FHV(Maeda et al., 1992), equine herpesvirus type 1 (EHV1) (Whalley et al.,19989), PRV (Robbins et al., 1987), BHV1 (Whitbeck et al., 1988), MDV(Ross et al., 1989) and HCMV (Kouzarides et al., 1987) are also cleaved.Furthermore, a sequence, Arg-X-Arg-Arg/Lys--Ser/Ala, similar to thesequence at the VZV cleavage site, Arg-Thr-Arg-Arg--Ser, is present atvirtually the same location in each of these gB glycoptoteins.Conversely, this sequence is not found in the HSV1 (Bzik et al., 1984)and EBV (Pellett et al., 1985) gB glycoproteins, which are not cleaved.The significance of this cleavage event is unknown. It does not appear,however, to be essential for replication, in vitro, since strains ofBHV1 (Blewett & Misra, 1991) and HCMV (Spaete et al., 1990) that havebeen mutated at the cleavage site, and therefore encode an uncleaved gBglycoprotein, are still infectious. Without wishing to be bound by thetheory that CHV gB is cleaved internally, proteolytically, the sequence,Arg-Lys-Arg-Arg--Ser, is present at the same location in CHV as in VZV,FHV, EHV1, PRV, BHV1, MDV and HCMV.

Example 4 Identification and Sequencing of the CHV gC Gene

CHV genomic fragments were randomly cloned into pBluescriptSK. Thenucleotide sequence of the termini of these fragments was determined andthe predicted amino acid sequence of potential ORFs were analyzed forhomology against the SWISS-PROT (Release 20) amino acid database. Usingthis methodology, a 12 kb XbaI fragment encoding an ORF with homology toherpesvirus gC glycoproteins was identified. The nucleotide sequence ofthis ORF is presented in FIG. 4. FIG. 4 shows the nucleotide sequenceand predicted amino acid sequence of the CHV gC homologue and ORF2. Theputative transmembrane region and potential TATA, CAAT andpolyadenylation signal sequences are underlined. Nucleotides andpredicted amino acid residues are numbered to the right of the sequence.The putative CHV gC gene starts at position 201 and ends at position1580. The predicted translation product is 459 amino acids long.Comparison of this amino acid sequence with the sequence of gCglycoproteins from other herpesviruses is shown in Table 2, below, andrevealed extensive homology, indicating that this ORF encodes the CHV gChomologue (Table 2).

                                      TABLE 2                                     __________________________________________________________________________    HOMOLOGY BETWEEN THE PREDICTED AMINO ACID                                     SEQUENCES OF 9 HERPESVIRUS gC GLYCOPROTEINS                                   FHV      EHV1 EHV4 PRV BHV1 VZV MDV HSV1                                      __________________________________________________________________________    CHV  44  32   34   27  27   29  27  25                                        FHV      32   33   29  31   28  25  23                                        EHV1          81   31  32   30  27  27                                        EHV4               32  31   31  25  27                                        PRV                    37   27  25  29                                        BHV1                        29  25  27                                        VzV                             22  22                                        MDV                                 23                                        __________________________________________________________________________

Values in Table 2 were obtained using the ALIGN Plus program and areexpressed as percent homology. The entire gC amino acid sequence wasused. See Table 1 for alignment parameters. References: FHV (Audonnet,unpublished results), EHV1 (Allen & Coogle, 1988), EHV4 (Nicolson &Onions, 1990), PRV (Robbins et al., 1986), BHV1 (Fitzpatrick et al.,1989), VZV (Davison & Scott, 1986), MDV (Ihara et al., 1989) and HSV1(McGeoch et al., 1988).

Example 5 Analysis of the CHV gC Nucleotide Sequence

Potential TATA box sequences (TATA) are found at positions 22 and 81,approximately 180 bp and 120 bp upstream from the CHV gC initiationcodon (FIG. 4). An additional TATA sequence is found at position 175.Due to its proximity to the gC initiation codon, however, this sequencemay not be a potential TATA box sequence. Potential CAAT box sequences(CAAT and ATTG) are found at positions 13, 59 and 119, approximately 190bp, 140 bp and 80 bp upstream from the gC initiation codon. A potentialpolyadenylation signal sequence (AATAAA) is found at position 1744,approximately 165 bp downstream from the CHV gC termination codon and 45bp within ORF2 (see below). Other potential polyadenylation signal-likesequences are also found in the gC 3'-noncoding region.

Like the CHV gB gene, the nucleotide sequence surrounding the CHV gCinitiation codon (AAAATGA) is favorable with respect to Kozak's rules atposition -3, but not at position +4 (FIG. 4). The FHV (Audonnet,unpublished results), EHV1 (Allen & Coogle, 1988) and VZV (Davison &Scott, 1986) gC genes also contain an unfavorable nucleotide at position+4.

Example 6 Analysis of the CHV gC Amino Acid Sequence Predicted

The deduced amino acid sequence of the CHV gC homologue is presented inFIG. 4. FIG. 5 shows the hydropathicity analysis of the CHV gChomologue. The profile was obtained with the PC/GENE SOAP program, usingthe method of Kyte & Doolittle (1982) and an interval of 13 amino acids.The vertical axis represents relative hydropathicity, where positivevalues are hydrophobic and negative values are hydrophilic. Thehorizontal axis represents the amino acid number of the CHV gChomologue.

Hydropathicity analysis of the predicted CHV gC amino acid sequencerevealed the presence of 2 prominent hydrophobic peaks (FIG. 5). Thefirst peak, located at the N-terminus, without wishing to be bound byany one theory, represents a potential signal sequence. Althoughanalysis with PSIGNAL does not identify the N-terminal end of thispolypeptide as a potential signal sequence, this region does have abasic N-terminal region, a hydrophobic core (residues 6-20) and arelatively polar C-terminal region (FIG. 4). The second hydrophobicpeak, with a predicted membrane-spanning segment between residues424-433 and 449-456 (using the method of Klein et al. (1985)), withoutwishing to be bound by any one theory, functions as a membrane anchorregion. FIG. 6 shows the amino acid homology of 4 gC homologues. Theamino acid sequences of the CHV, FHV, EHV1 and HSV1 gC homologues (forreferences see Table 2) were aligned using the PC/GENE CLUSTAL program.Gaps, indicated by dashes, were introduced to maximize homology. Alignedresidues which are identical in all 4 sequences are indicated by anasterisk (*). Aligned residues which are identical in the majority ofsequences are indicated by a period (.). Conserved cysteine residues areboxed. Potential N-linked glycosylation sites are shaded.

Alignment of the CHV gC amino acid sequence with homologous sequencesfrom other herpesviruses revealed a moderate level of homologythroughout the entire sequence, with the exception of the N-terminus(FIG. 6). This alignment also revealed that the majority of cysteineresidues are perfectly conserved. For example, CHV gC contains 10cysteine residues, 8 of which are perfectly conserved in allalpha-herpesviruses. In fact, the only cysteine residues in CHV gC thatare not conserved are located in the putative transmembrane orintracellular domains. These results show that the gC glycoproteins haverelatively similar tertiary structures. Alignment with other gCsequences also revealed the relative conservation of potential N-linkedglycosylation sites.

Example 7 Identification and Sequencing of ORF2

Nucleotide sequence analysis of the region downstream from the CHV gCgene revealed the presence of a second ORF (FIG. 4). This ORF (ORF2)starts at position 1699 and ends at position 2226. The predictedtranslation product is 175 amino acids long. Table 3, below, shows thecomparison of this amino acid sequence with the SWISS-PROT (Release 23)database revealed significant homology with the ORFs located downstreamfrom other alpha-herpesvirus gC genes, The homology scores for the ORF2homologues shows that in CHV, FHV, EHV1, equine herpesvirus type 4(EHV4), MDV, herpesvirus of turkey (HVT) and possibly HSV1, the ORFlocated downstream from the gC gene represents a highly divergent, butevolutionarily related, gene family. Conversely, the ORF (gene 13)located next to the VZV gC gene does not exhibit significant homologywith any of the other comparably positioned ORFs. Furthermore, gene 13is oriented on the genome in the opposite direction relative to all theother ORF2-like genes (Davison & Scott, 1986). These results areconsistent with the proposed functions of the proteins encoded by these2 groups of genes; VZV gene 13 encodes a thymidylate synthetase (Davisonand Scott, 1986), whereas the HSV1 ORF2-like gene (UL45) encodes aputative virion protein (Telford et al., 1992). Therefore, the ORFslocated next to the gC gene in CHV, FHV, EHV1, EHV4, MDV, HVT andpossibly HSV1 encode proteins that are structurally and functionallyunrelated to the protein encoded downstream from the VZV gC homologue.

                  TABLE 3                                                         ______________________________________                                        HOMOLOGY BETWEEN THE PREDICTED AMINO ACID                                     SEQUENCES OF THE ORFS LOCATED ADJACENT TO THE gC                              GENE IN 8 HERPESVIRUSES                                                       FHV       EHV1    EHV4    MDV   HTV   HSV1  VZV                               ______________________________________                                        CHV   197(22) 211(22) 219(21)                                                                              62(4)                                                                              105(13)                                                                             53(4) 31(0)                           FHV           177(24) 167(18)                                                                              69(4)                                                                               66(4)                                                                              40(1) 52(1)                           EHV1                  470(50)                                                                              95(8)                                                                              104(9)                                                                              79(7) 58(3)                           EHV4                        132(8)                                                                              130(11)                                                                             60(5) 30(0)                           MDV                               767(75)                                                                             83(6) 28(0)                           HTV                                     91(7) 33(0)                           HSV1                                          49(2)                           ______________________________________                                    

Values in Table 3 were obtained using the FASTA and RDF2 programs(Pearson & Lipman, 1988). A ktup of 1 was used. Values in parenthesesrepresent the number of standard deviations between the FASTA score andthe mean of the scores obtained from 100 randomly permutated versions ofthe potentially related sequence. References: FHV (Audonnet, unpublishedresults), EHV1 (Telford et al., 1992), EHV4 (Nicolson & Onions, 1990),MDV (Ihara et al., 1989), HVT (Kato et al., 1989), HSV1 (McGeoch et al.,1988) and VZV (Davison & Scott, 1986).

Example 8 Analysis of the CHV ORF2 Nucleotide Sequence

Potential TATA box sequences (TATA) are found at positions 1604, 1606,1635 and 1662, approximately 95, 93, 65 and 35 bp upstream from the ORF2initiation codon and approximately 24, 26, 55 and 80 bp downstream fromthe gC gene termination codon (FIG. 4). A potential CAAT box sequence(CAAT) is found at position 1584, approximately 115 bp upstream from theinitiation codon. Potential polyadenylation signal sequences (AATAAA)are found at overlapping positions 2225, 2229, 2234 and 2238,approximately 0-15 bp downstream from the ORF2 termination codon. Thenucleotide sequence surrounding the ORF2 initiation codon (AATATGG) isfavorable with respect to Kozak's rules at positions -3 and +4.

Example 9 Identification and Sequencing of the CHV gD Gene

Employing the same methodology used to map the CHV gC homologue, a 7 kbPstI fragment encoding an ORF with homology to herpesvirus gDglycoproteins was identified. FIG. 7 shows the nucleotide sequence andpredicted amino acid sequence of the CHV gD homologue. The putativesignal sequence, transmembrane region and potential polyadenylationsignal sequences are underlined. Nucleotides and predicted amino acidresidues are numbered to the right of the sequence. The CHV gD genestarts at position 201 and ends at position 1238. The translationproduct (predicted) is 345 amino acids long. Table 4, below, providescomparison of this amino acid sequence with the sequence of other gDglycoproteins and, revealed extensive homology, indicating that this ORFencodes the CHV gD homologue.

                  TABLE 4                                                         ______________________________________                                        Homology between the predicted amino acid                                     sequences of 6 herpesvirus gD glycoproteins                                   FHV          EHV1    PRV      BHV1  HSV1                                      ______________________________________                                        CHV     45       35      27     34    21                                      FHV              31      30     34    24                                      EHV1                     26     27    21                                      PRV                             37    27                                      BHV1                                  24                                      ______________________________________                                    

Values in Table 4 were obtained using the ALIGN Plus program and areexpressed as percent homology. The entire gD amino acid sequence wasused. See Table 1 for alignment parameters. References: FHV (Audonnet,unpublished results), EHV1 (Flowers et al., 1991), PRV (Petrovskis etal., 1986), BHV1 (Tikoo et al., 1990) and HSV1 (Lasky & Dowbenko, 1984).

Example 10 Analysis of the CHV gD Nucleotide Sequence

No TATA or CAAT/ATTG sequences were identified immediately upstream fromthe CHV gD gene (FIG. 7). Numerous potential TATA box-like sequences,however, were found. Potential polyadehylation signal sequences (AATAAA)were found at positions 1260 and 1287, approximately 25 bp and 50 bpdownstream from the CHV gD termination codon. Like the CHV gB and gCgenes, the nucleotide sequence surrounding the CHV gD initiation codon(AAAATGA) is favorable with respect to Kozak's rules at position -3, butnot at position +4 (FIG. 7). The FHV (Audonnet, unpublished results),EHV1 (Audonnet et al., 1990; Flowers et al., 1991), PRV (Petrovskis etal., 1986) and BHV1 (Tikoo et al., 1990) gD genes also contain anunfavorable nucleotide at position +4.

Example 11 Analysis of the Predicted CHV gD Amino Acid Sequence

The deduced amino acid sequence of the CHV gD homologue is presented inFIG. 7. FIG. 8 shows the hydropathicity analysis of the CHV gDhomologue. The profile was obtained with the PC/GENE SOAP program, usingthe method of Kyte & Doolittle (1982) and an interval of 11 amino acids.The vertical axis represents relative hydropathicity, where positivevalues are hydrophobic and negative values are hydrophilic. Thehorizontal axis represents the amino acid number of the CHV gDhomologue.

Hydropathicity analysis of the predicted CHV gD amino acid sequencerevealed the presence of 2 prominent hydrophobic peaks (FIG. 8). Thefirst peak, located at the N-terminus, without wishing to be bound byany one theory, represents a potential signal sequence. In fact, PSIGNALidentified a potential cleavage site between positions 16 and 17. Thesecond hydrophobic peak, with a predicted membrane-spanning segmentbetween residues 304-311 and 327-332 (using the method of Klein et al.(1985)), without wishing to be bound by any one theory, functions as amembrane anchor region.

FIG. 9 shows amino acid homology of 4 gD homologues. The amino acidsequences of the CHV, FHV, EHV1 and HSV1 gD homologues (for referencessee Table 4) were aligned using the PC/GENE CLUSTAL program. Gaps,indicated by dashes, were introduced to maximize homology. Alignedresidues which are identical in all 4 sequences are indicated by anasterisk (*). Aligned residues which are identical in the majority ofsequences are indicated by a period (.). Conserved cysteine residues areboxed. Potential N-linked glycosylation sites are shaded. Alignment ofthe CHV gD amino acid sequence with homologous sequences from otherherpesviruses revealed a moderate level of homology throughout theentire sequence, with the exception of the N-terminus (FIG. 9). Thisalignment also revealed that the vast majority of cysteine residues areperfectly conserved. For example, CHV gD contains 6 cysteine residues,all of which are perfectly conserved in all alpha-herpesviruses. Theseresults show that the gD glycoproteins have relatively similar tertiarystructures. This alignment also revealed that the potential N-linkedglycosylation sites are well conserved. Without wishing to be bound byany theory that the CHV gD glycosylation sites are utilized, it is knownthat all of the potential HSV1 gD glycosylation sites are used (Sodoraet al., 1991).

Example 12 Genomic Organization

The gB, gC and gD genes were not mapped to specific locations on the CHVgenome. Nucleotide sequence analyses of the regions flanking thesegenes, however, indicates that the genomic organization of CHV issimilar to other alpha-herpesviruses. For example, the ORF locatedimmediately upstream from the CHV gB gene has homology with gene 30 ofVZV (Davison & Scott, 1986) and UL28 of HSV1 (McGeoch et al., 1988),both of which are located immediately upstream from the gB homologue inthose viruses. ORF2, located immediately downstream from the CHV gCgene, has homology with the ORFs located immediately downstream from thegC homologue in FHV (Audonnet, unpublished results), EHV1 (Telford etal., 1992), EHV4 (Nicolson & Onions, 1990), HVT (Kato et al., 1988) andperhaps HSV1 (McGeoch et al., 1988. Additionally, the ORF locatedimmediately downstream from the CHV gD gene has homology to the gI geneof EHV1 (Audonnet et al., 1990) and the gp63 gene of PRV (Petrovskis etal., 1986), both of which are located immediately downstream from the gDhomologue in those viruses (data not shown).

Example 13 Construction of Plasmid pSD460 for Deletion of ThymidineKinase Gene (J2R)

Referring now to FIG. 10, plasmid pSD406 contains vaccinia HindIII J(pos. 83359-88377) cloned into pUC8. pSD406 was cut with HindIII andPvuII, and the 1.7 kb fragment from the left side of HindIII J clonedinto PUC8 cut with HindIII/SmaI, forming pSD447. pSD447 contains theentire gene for J2R (pos. 83855-84385). The initiation codon iscontained within an NlaIII site and the termination codon is containedwithin an SspI site. Direction of transcription is indicated by an arrowin FIG. 10.

To obtain a left flanking arm, a 0.8 kb HindIII/EcoRI fragment wasisolated from pSD447, then digested with NlaIII and a 0.5 kbHindIII/NlaIII fragment isolated. Annealed synthetic oligonucleotidesMPSYN43/MPSYN44 (SEQ ID NO:24/SEQ ID NO:25)

                                   SmaI                                           MPSYN43   5'     TAATTAACTAGCTACCCGGG     3'                                  MPSYN44   3' GTACATTAATTGATCGATGGGCCCTTAA 5'                                              NlaIII                  EcoRI                                 

were ligated with the 0.5 kb HindIII/NlaIII fragment into pUC18 vectorplasmid cut with HindIII/EcoRI, generating plasmid pSD449.

To obtain a restriction fragment containing a vaccinia right flankingarm and pUC vector sequences, pSD447 was cut with SspI (partial) withinvaccinia sequences and HindIII at the pUC/vaccinia junction, and a 2.9kb vector fragment isolated. This vector fragment was ligated withannealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID NO:26/SEQ IDNO:27)

              HindIII SmaI                                                        MPSYN45 5'  AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA                            MPSYN46 3'      AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT                                                   NotI              SspI                                          ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT  3' MPSYN45                               TGCTAGACATCAATCGCCGGCGGATTAATTGATTA  5' MPSYN46                  

generating pSD459.

To combine the left and right flanking arms into one plasmid, a 0.5 kbHindIII/SmaI fragment was isolated from pSD449 and ligated with pSD459vector plasmid cut with HindIII/SmaI, generating plasmid pSD460. pSD460was used as donor plasmid for recombination with wild type parentalvaccinia virus Copenhagen strain VC-2. ³² P labelled probe wassynthesized by primer extension using MPSYN45 (SEQ ID NO:26) as templateand the complementary 20 mer oligonucleotide MPSYN47 (SEQ ID NO:28) (5'TTAGTTAATTAGGCGGCCGC 3') as primer. Recombinant virus vP410 wasidentified by plaque hybridization.

Example 14 Construction of Plasmid pSD486 for Deletion of HemorrhagicRegion (B13R+B14R)

Referring now to FIG. 11, plasmid pSD419 contains vaccinia SalI G (pos.160,744-173,351) cloned into pUC8. pSD422 contains the contiguousvaccinia SalI fragment to the right, SalI J (pos. 173,351-182,746)cloned into pUC8. To construct a plasmid deleted for the hemorrhagicregion, u, B13R-B14R (pos. 172,549-173,552), pSD419 was used as thesource for the left flanking arm and pSD422 was used as the source ofthe right flanking arm. The direction of transcription for the u regionis indicated by an arrow in FIG. 11.

To remove unwanted sequences from pSD419, sequences to the left of theNcoI site (pos. 172,253) were removed by digestion of pSD419 withNcoI/SmaI followed by blunt ending with Klenow fragment of E. colipolymerase and ligation generating plasmid pSD476. A vaccinia rightflanking arm was obtained by digestion of pSD422 with HpaI at thetermination codon of B14R and by digestion with NruI 0.3 kb to theright. This 0.3 kb fragment was isolated and ligated with a 3.4 kbHincII vector fragment isolated from pSD476, generating plasmid pSD477.The location of the partial deletion of the vaccinia u region in pSD477is indicated by a triangle. The remaining B13R coding sequences inpSD477 were removed by digestion with ClaI/HpaI, and the resultingvector fragment was ligated with annealed synthetic oligonucleotidesSD22mer/SD20mer (SEQ ID NO:29/SEQ ID NO:30)

                   ClaI         BamHI HpaI                                        SD22mer     5' CGATTACTATGAAGGATCCGTT  3'                                     SD20mer     3'   TAATGATACTTCCTAGGCAA  5'                                 

generating pSD479. pSD479 contains an initiation codon (underlined)followed by a BamHI site. To place E. coli Beta-galactosidase in theB13-B14 (u) deletion locus under the control of the u promoter, a 3.2 kbBamHI fragment containing the Beta-galactosidase gene (Shapira et al.,1983) was inserted into the BamHI site of pSD479, generating pSD479BG.pSD479BG was used as donor plasmid for recombination with vaccinia virusvP410. Recombinant vaccinia virus vP533 was isolated as a blue plaque inthe presence of chromogenic substrate X-gal. In vP533 the B13R-B14Rregion is deleted and is replaced by Beta-galactosidase.

To remove Beta-galactosidase sequences from vP533, plasmid pSD486, aderivative of pSD477 containing a polylinker region but no initiationcodon at the u deletion junction, was utilized. First the ClaI/HpaIvector fragment from pSD477 referred to above was ligated with annealedsynthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:31/SEQ ID NO:32)

                 ClaI          SacI        XhoI        HpaI                       SD42mer   5' CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT  3'                    SD40mer   3'   TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA  5'                                         BglII       SmaI        BamHI                        

generating plasmid pSD478. Next the EcoRI site at the pUC/vacciniajunction was destroyed by digestion of pSD478 with EcoRI followed byblunt ending with Klenow fragment of E. coli polymerase and ligation,generating plasmid pSD478E⁻ .pSD478E⁻ was digested with BamHI and HpaIand ligated with annealed synthetic oligonucleotides HEM5/HEM6 (SEQ IDNO:33/SEQ ID NO:34)

                    BamHI EcoRI   HpaI                                            HEM5          5'  GATCCGAATTCTAGCT 3'                                         HEM6          3'      GCTTAAGATCGA 5'                                     

generating plasmid pSD486. pSD486 was used as donor plasmid forrecombination with recombinant vaccinia virus vP533, generating vP553,which was isolated as a clear plaque in the presence of X-gal.

Example 15 Construction of Plasmid pMP494Δ for Deletion of ATI Region(A26L)

Referring now to FIG. 12, pSD414 contains SalI B cloned into pUC8. Toremove unwanted DNA sequences to the left of the A26L region, pSD414 wascut with XbaI within vaccinia sequences (pos. 137,079) and with HindIIIat the pUC/vaccinia junction, then blunt ended with Klenow fragment ofE. coli polymerase and ligated, resulting in plasmid pSD483. To removeunwanted vaccinia DNA sequences to the right of the A26L region, pSD483was cut with EcoRI (pos. 140,665 and at the pUC/vaccinia junction) andligated, forming plasmid pSD484. To remove the A26L coding region,pSD484 was cut with NdeI (partial) slightly upstream from the A26L ORF(pos. 139,004) and with HpaI (pos. 137,889) slightly downstream from theA26L ORF. The 5.2 kb vector fragment was isolated and ligated withannealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:35/SEQ IDNO:36)

           NdeI                                                                   ATI3 5' TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT                   ATI4 3'   ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAgTTTTTTATTCA                                BglII EcoRI HpaI                                                      TATATAAATAGATCTGAATTCGTT 3' ATI3                                              ATATATTTATCTAGACTTAAGCAA 5' ATI4                                     

reconstructing the region upstream from A26L and replacing the A26L ORFwith a short polylinker region containing the restriction sites BglII,EcoRI and HpaI, as indicated above. The resulting plasmid was designatedpSD485. Since the BglII and EcoRI sites in the polylinker region ofpSD485 are not unique, unwanted BglII and EcoRI sites were removed fromplasmid pSD483 (described above) by digestion with BglII (pos. 140,136)and with EcoRI at the pUC/vaccinia junction, followed by blunt endingwith Klenow fragment of E. coli polymerase and ligation. The resultingplasmid was designated pSD489. The 1.8 kb ClaI (pos. 137,198)/EcoRV(pos. 139,048) fragment from pSD489 containing the A26L ORF was replacedwith the corresponding 0.7 kb polylinker-containing ClaI/EcoRV fragmentfrom pSD485, generating pSD492. The BglII and EcoRI sites in thepolylinker region of pSD492 are unique.

A 3.3 kb BglII cassette containing the E. coli Beta-galactosidase gene(Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter(Bertholet et al., 1985; Perkus et al., 1990) was inserted into theBglII site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used inrecombination with rescuing virus vP553. Recombinant vaccinia virus,vP581, containing Beta-galactosidase in the A26L deletion region, wasisolated as a blue plaque in the presence of X-gal.

To generate a plasmid for the removal of Beta-galactosidase sequencesfrom vaccinia recombinant virus vP581, the polylinker region of plasmidpSD492 was deleted by mutagenesis (Mandecki, 1986) using syntheticoligonucleotide MPSYN177 (SEQ ID NO:37) (5'AAAATGGGCGTGGATTGTTAACTTTATATAACTTATTTTTTGAATATAC 3'). In the resultingplasmid, pMP494Δ, vaccinia DNA encompassing positions [137,889-138,937],including the entire A26L ORF is deleted. Recombination between thepMP494Δ and the Beta-galactosidase containing vaccinia recombinant,vP581, resulted in vaccinia deletion mutant vP618, which was isolated asa clear plaque in the presence of X-gal.

Example 16 Construction of Plasmid pSD467 for Deletion of HemagglutininGene (A56R)

Referring now to FIG. 13, vaccinia SalI G restriction fragment (pos.160,744-173,351) crosses the HindIII A/B junction (pos. 162,539). pSD419contains vaccinia SalI G cloned into pUC8. The direction oftranscription for the hemagglutinin (HA) gene is indicated by an arrowin FIG. 13. Vaccinia sequences derived from HindIII B were removed bydigestion of pSD419 with HindIII within vaccinia sequences and at thepUC/vaccinia junction followed by ligation. The resulting plasmid,pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vacciniasequences to the left and 0.4 kb of vaccinia sequences to the right.A56R coding sequences were removed by cutting pSD456 with RsaI (partial;pos. 161,090) upstream from A56R coding sequences, and with EagI (pos.162,054) near the end of the gene. The 3.6 kb RsaI/EagI vector fragmentfrom pSD456 was isolated and ligated with annealed syntheticoligonucleotides MPSYN59 (SEQ ID NO:38), MPSYN62 (SEQ ID NO:39), MPSYN60(SEQ ID NO:40), and MPSYN61 (SEQ ID NO:41)

                 RsaI                                                             MPSYN59    5' ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT-                      MPSYN62    3' TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA-                      MPSYN59      AGTTGATAGAACAAAATACATAATTT 3'                                    MPSYN62      TCAACTATCT 5'                                                    MPSYN60    5'                 TGTAAAAATAAATCACTTTTTATA-                       MPSYN61    3' TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT-                                       BglII SmaI  PstI  EagI                                        MPSYN60      CTAAGATCTCCCGGGCTGCAGC     3'                                    MPSYN61      GATTCTAGAGGGCCCGACGTCGCCGG 5'                                

reconstructing the DNA sequences upstream from the A56R ORF andreplacing the A56R ORF with a polylinker region as indicated above. Theresulting plasmid is pSD466. The vaccinia deletion in pSD466 encompassespositions [161,185-162,053]. The site of the deletion in pSD466 isindicated by a triangle in FIG. 13.

A 3.2 kb BglII/BamHI (partial) cassette containing the E. coliBeta-galactosidase gene (Shapira et al., 1983) under the control of thevaccinia 11 kDa promoter (Bertholet et al., 1985; Guo et al., 1989) wasinserted into the BglII site of pSD466, forming pSD466KBG. PlasmidpSD466KBG was used in recombination with rescuing virus vP618.Recombinant vaccinia virus, vP708, containing Beta-galactosidase in theA56R deletion, was isolated as a blue plaque in the presence of X-gal.

Beta-galactosidase sequences were deleted from vP708 using donor plasmidpSD467. pSD467 is identical to pSD466, except that EcoRI, SmaI and BamHIsites were removed from the pUC/vaccinia junction by digestion of pSD466with EcoRI/BamHI followed by blunt ending with Klenow fragment of E.coli polymerase and ligation. Recombination between vP708 and pSD467resulted in recombinant vaccinia deletion mutant, vP723, which wasisolated as a clear plaque in the presence of X-gal.

Example 17 Construction of Plasmid pMPCSK1Δ for Deletion of Open ReadingFrames [C7L-K1L]

Referring now to FIG. 14, the following vaccinia clones were utilized inthe construction of pMPCSK1Δ. pSD420 is SalI H cloned into pUC8. pSD435is KpnI F cloned into pUC18. pSD435 was cut with SphI and religated,forming pSD451. In pSD451, DNA sequences to the left of the SphI site(pos. 27,416) in HindIII M are removed (Perkus et al., 1990). pSD409 isHindIII M cloned into pUC8.

To provide a substrate for the deletion of the [C7L-K1L] gene clusterfrom vaccinia, E. coli Beta-galactosidase was first inserted into thevaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminatethe BglII site in pSD409, the plasmid was cut with BglII in vacciniasequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction,then ligated to form plasmid pMP409B. pMP409B was cut at the unique SphIsite (pos. 27,416). M2L coding sequences were removed by mutagenesis(Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide

                                              BglII                               MPSYN82 (SEQ ID NO:47)                                                                        5' TTTCTGTATATTTGCACCAATTTAGATCTT-                                          ACTCAAAATATGTAACAATA 3'                                     

The resulting plasmid, pMP409D, contains a unique BglII site insertedinto the M2L deletion locus as indicated above. A 3.2 kb BamHI(partial)/BglII cassette containing the E. coli Beta-galactosidase gene(Shapira et al., 1983) under the control of the 11 kDa promoter(Bertholet et al., 1985) was inserted into pMP409D cut with BglII. Theresulting plasmid, pMP409DBG (Guo et al., 1990), was used as donorplasmid for recombination with rescuing vaccinia virus vP723.Recombinant vaccinia virus, vP784, containing Beta-galactosidaseinserted into the M2L deletion locus, was isolated as a blue plaque inthe presence of X-gal.

A plasmid deleted for vaccinia genes [C7L-K1L] was assembled in pUC8 cutwith SmaI, HindIII and blunt ended with Klenow fragment of E. colipolymerase. The left flanking arm consisting of vaccinia HindIII Csequences was obtained by digestion of pSD420 with XbaI (pos. 18,628)followed by blunt ending with Klenow fragment of E. coli polymerase anddigestion with BglII (pos. 19,706). The right flanking arm consisting ofvaccinia HindIII K sequences was obtained by digestion of pSD451 withBglII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid,pMP581CK is deleted for vaccinia sequences between the BglII site (pos.19,706) in HindIII C and the BglII site (pos. 29,062) in HindIII K. Thesite of the deletion of vaccinia sequences in plasmid pMP581CK isindicated by a triangle in FIG. 14.

To remove excess DNA at the vaccinia deletion junction, plasmidpMP581CK, was cut at the NcoI sites within vaccinia sequences (pos.18,811; 19,655), treated with Bal-31 exonuclease and subjected tomutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233(SEQ ID NO:43) 5'-TGTCATTTAACACTATACTCATATTAATAAAAATAATATTTATT-3'. Theresulting plasmid, pMPCSK1Δ, is deleted for vaccinia sequences positions18,805-29,108, encompassing 12 vaccinia open reading frames [C7L-K1L].Recombination between pMPCSK1Δ and the Beta-galactosidase containingvaccinia recombinant, vP784, resulted in vaccinia deletion mutant,vP804, which was isolated as a clear plaque in the presence of X-gal.

Example 18 Construction of Plasmid pSD548 for Deletion of Large Subunit,Ribonucleotide Reductase (I4L)

Referring now to FIG. 15, plasmid pSD405 contains vaccinia HindIII I(pos. 63,875-70,367) cloned in pUC8. pSD405 was digested with EcoRVwithin vaccinia sequences (pos. 67,933) and with SmaI at thepUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 wasused as the source of all the vaccinia restriction fragments used in theconstruction of pSD548.

The vaccinia I4L gene extends from position 67,371-65,059. Direction oftranscription for I4L is indicated by an arrow in FIG. 15. To obtain avector plasmid fragment deleted for a portion of the I4L codingsequences, pSD518 was digested with BamHI (pos. 65,381) and HpaI (pos.67,001) and blunt ended using Klenow fragment of E. coli polymerase.This 4.8 kb vector fragment was ligated with a 3.2 kb SmaI cassettecontaining the E. coli Beta-galactosidase gene (Shapira et al., 1983)under the control of the vaccinia 11 kDa promoter (Bertholet et al.,1985; Perkus et al., 1990), resulting in plasmid pSD524KBG. pSD524KBGwas used as donor plasmid for recombination with vaccinia virus vP804.Recombinant vaccinia virus, vP855, containing Beta-galactosidase in apartial deletion of the I4L gene, was isolated as a blue plaque in thepresence of X-gal.

To delete Beta-galactosidase and the remainder of the I4L ORF fromvP855, deletion plasmid pSD548 was constructed. The left and rightvaccinia flanking arms were assembled separately in pUC8 as detailedbelow and presented schematically in FIG. 15.

To construct a vector plasmid to accept the left vaccinia flanking arm,pUC8 was cut with BamHI/EcoRI and ligated with annealed syntheticoligonucleotides 518A1/518A2 (SEQ ID NO:44/SEQ ID NO:45)

             BamHI   RsaI                                                         518A1 5' GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAT                   518A2 3'     GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA                                          BglII    EcoRI                                                    TTGAGAATAAAAAGATCTTAGG     3'   518A1                                         AACTCTTATTTTTCTAGAATCCTTAA 5'   518A2                          

forming plasmid pSD531. pSD531 was cut with RsaI (partial) and BamHI anda 2.7 kb vector fragment isolated. pSD518 was cut with BglII (pos.64,459)/RsaI (pos. 64,994) and a 0.5 kb fragment isolated. The twofragments were ligated together, forming pSD537, which contains thecomplete vaccinia flanking arm left of the I4L coding sequences.

To construct a vector plasmid to accept the right vaccinia flanking arm,pUC8 was cut with BamHI/EcoRI and ligated with annealed syntheticoligonucleotides 518B1/518B2 (SEQ ID NO:46/SEQ ID NO:47)

               BamHI BglII SmaI                                                   518B1   5'  GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAG-                   518B2   3'      GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATC-                                          RsaI   EcoRI                                                GGATTTGACGTATGTAGCGTACTAGG     3'   518B1                                     CCTAAACTGCATACTACGCATGATCCTTAA 5'   518B2                            

forming plasmid pSD532. pSD532 was cut with RsaI (partial)/EcoRI and a2.7 kb vector fragment isolated. pSD518 was cut with RsaI withinvaccinia sequences (pos. 67,436) and EcoRI at the vaccinia/pUC junction,and a 0.6 kb fragment isolated. The two fragments were ligated together,forming pSD538, which contains the complete vaccinia flanking arm to theright of I4L coding sequences.

The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/BglIIfragment from pSD538 and ligated into pSD537 vector plasmid cut withEcoRI/BglII. In the resulting plasmid, pSD539, the I4L ORF (pos.65,047-67,386) is replaced by a polylinker region, which is flanked by0.6 kb vaccinia DNA to the left and 0.6 kb vaccinia DNA to the right,all in a pUC background. The site of deletion within vaccinia sequencesis indicated by a triangle in FIG. 15. To avoid possible recombinationof Beta-galactosidase sequences in the pUC-derived portion of pSD539with Beta-galactosidase sequences in recombinant vaccinia virus vP855,the vaccinia I4L deletion cassette was moved from pSD539 into pRC11, apUC derivative from which all Beta-galactosidase sequences have beenremoved and replaced with a polylinker region (Colinas et al., 1990).pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment isolated. Thisfragment was ligated into pRC11 cut with EcoRI/PstI (2.35 kb), formingpSD548. Recombination between pSD548 and the Beta-galactosidasecontaining vaccinia recombinant, vP855, resulted in vaccinia deletionmutant vP866, which was isolated as a clear plaque in the presence ofX-gal.

DNA from recombinant vaccinia virus vP866 was analyzed by restrictiondigests followed by electrophoresis on an agarose gel. The restrictionpatterns were as expected. Polymerase chain reactions (PCR) (Engelke etal., 1988) using vP866 as template and primers flanking the six deletionloci detailed above produced DNA fragments of the expected sizes.Sequence analysis of the PCR generated fragments around the areas of thedeletion junctions confirmed that the junctions were as expected.Recombinant vaccinia virus vP866, containing the six engineereddeletions as described above, was designated vaccinia vaccine strain"NYVAC."

Example 19 Insertion of a Rabies Glycoprotein G Gene into NYVAC

The gene encoding rabies glycoprotein G under the control of thevaccinia H6 promoter (Taylor et al., 1988a,b) was inserted into TKdeletion plasmid pSD513. pSD513 is identical to plasmid pSD460 (FIG. 10)except for the presence of a polylinker region.

Referring now to FIG. 16, the polylinker region was inserted by cuttingpSD460 with SmaI and ligating the plasmid vector with annealed syntheticoligonucleotides VQ1A/VQ1B (SEQ ID NO:48/SEQ ID NO:49)

           SmaI BglII XhoI  PstI  NarI  BamHI                                     VQ1A 5'  GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT 3'                          VQ1B 3'  CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA 5'                      

to form vector plasmid pSD513. pSD513 was cut with SmaI and ligated witha SmaI ended 1.8 kb cassette containing the gene encoding the rabiesglycoprotein G gene under the control of the vaccinia H6 promoter(Taylor et al., 1988a,b). The resulting plasmid was designated pRW842.pRW842 was used as donor plasmid for recombination with NYVAC rescuingvirus (vP866). Recombinant vaccinia virus vP879 was identified by plaquehybridization using ³² P-labelled DNA probe to rabies glycoprotein Gcoding sequences.

The modified recombinant viruses of the present invention provideadvantages as recombinant vaccine vectors. The attenuated virulence ofthe vector advantageously reduces the opportunity for the possibility ofa runaway infection due to vaccination in the vaccinated individual andalso diminishes transmission from vaccinated to unvaccinated individualsor contamination of the environment.

The modified recombinant viruses are also advantageously used in amethod for expressing a gene product in a cell cultured in vitro byproducing into the cell the modified recombinant virus having foreignDNA which codes for and expresses gene products in the cell.

Example 20 Construction of TROVAC-NDV Expressing the Fusion andHemagglutinin-neuraminidase Glycoproteins of Newcastle Disease Virus

This example describes the development of TROVAC, a fowlpox virus vectorand, of a fowlpox Newcastle Disease Virus recombinant designatedTROVAC-NDV and its safety and efficacy. A fowlpox virus (FPV) vectorexpressing both F and HN genes of the virulent NDV strain Texas wasconstructed. The recombinant produced was designated TROVAC-NDV.TROVAC-NDV expresses authentically processed NDV glycoproteins in aviancells infected with the recombinant virus and inoculation of day oldchicks protects against subsequent virulent NDV challenge.

Cells and Viruses.

The Texas strain of NDV is a velogenic strain. Preparation of cDNAclones of the F and HN genes has been previously described (Taylor etal., 1990; Edbauer et al., 1990). The strain of FPV designated FP-1 hasbeen described previously (Taylor et al., 1988a). It is a vaccine strainuseful in vaccination of day old chickens. The parental virus strainDuvette was obtained in France as a fowlpox scab from a chicken. Thevirus was attenuated by approximately 50 serial passages in chickenembryonated eggs followed by 25 passages on chicken embryo fibroblastcells. The virus was subjected to four successive plaque purifications.One plaque isolate was further amplified in primary CEF cells and astock virus, designated as TROVAC, established. The stock virus used inthe in vitro recombination test to produce TROVAC-NDV had been subjectedto twelve passages in primary CEF cells from the plaque isolate.

Construction of a Cassette for NDV-F.

A 1.8 kbp BamHI fragment containing all but 22 nucleotides from the 5'end of the F protein coding sequence was excised from pNDV81 (Taylor etal., 1990) and inserted at the BamHI site of pUC18 to form pCE13. Thevaccinia virus H6 promoter previously described (Taylor et al., 1988a,b;Guo et al., 1989; Perkus et al., 1989) was inserted into pCE13 bydigesting pCE13 with SalI, filling in the sticky ends with Klenowfragment of E. coli DNA polymerase and digesting with HindIII. AHindIII - EcoRV fragment containing the H6 promoter sequence was theninserted into pCE13 to form pCE38. A perfect 5' end was generated bydigesting pCE38 with KpnI and NruI and inserting the annealed andkinased oligonucleotides CE75 (SEQ ID NO:50) and CE76 (SEQ ID NO:51) togenerate pCE47.

CE75: CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGATCCCGGTAC

CE76: CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGATATCG.

In order to remove non-coding sequence from the 3' end of the NDV-F aSmaI to PstI fragment from pCE13 was inserted into the SmaI and PstIsites of pUC18 to form pCE23. The non-coding sequences were removed bysequential digestion of pCE23 with SacI, BamHI, Exonuclease III, SInuclease and EcoRI. The annealed and kinased oligonucleotides CE42 (SEQID NO:52) and CE43 (SEQ ID NO:53) were then inserted to form pCE29.

CE42: AATTCGAGCTCCCCGGG

CE43: CCCGGGGAGCTCG

The 3' end of the NDV-F sequence was then inserted into plasmid pCE20already containing the 5' end of NDV-F by cloning a PstI - SacI fragmentfrom pCE29 into the PstI and SacI sites of pCE20 to form pCE32.Generation of pCE20 has previously been described in Taylor et al.,1990.

In order to align the H6 promoter and NDV-F 5' sequences contained inpCE47 with the 3' NDV-F sequences contained in pCE32, a HindIII - PstIfragment of pCE47 was inserted into the HindIII and PstI sites of pCE32to form pCE49. The H6 promoted NDV-F sequences were then transferred tothe de-ORFed F8 locus (described below) by cloning a HindIII - NruIfragment from pCE49 into the HindIII and SmaI sites of pJCA002(described below) to form pCE54. Transcription stop signals wereinserted into pCE54 by digesting pCE54 with SacI, partially digestingwith BamHI and inserting the annealed and kinased oligonucleotides CE166(SEQ ID NO:54) and CE167 (SEQ ID NO:55) to generate pCE58.

CE166: CTTTTTATAAAAAGTTAACTACGTAG

CE167: GATCCTACGTAGTTAACTTTTTATAAAAAGAGCT

A perfect 3' end for NDV-F was obtained by using the polymerase chainreaction (PCR) with pCE54 as template and oligonucleotides CE182 (SEQ IDNO:56) and CE183 (SEQ ID NO:57) as primers.

CE182: CTTAACTCAGCTGACTATCC

CE183: TACGTAGTTAACTTTTTATAAAAATCATATTTTTGTAGTGGCTC

The PCR fragment was digested with PvuII and HpaI and cloned into pCE58that had been digested with HpaI and partially digested with PvuII. Theresulting plasmid was designated pCE64. Translation stop signals wereinserted by cloning a HindIII - HpaI fragment which contains thecomplete H6 promoter and F coding sequence from pCE64 into the HindIIIand HpaI sites of pRW846 to generate pCE71, the final cassette forNDV-F. Plasmid pRW846 is essentially equivalent to plasmid pJCA002(described below) but containing the H6 promoter and transcription andtranslation stop signals. Digestion of pRW846 with HindIII and HpaIeliminates the H6 promoter but leaves the stop signals intact.

Construction of Cassette for NDV-HN.

Construction of plasmid pRW802 was previously described in Edbauer etal., 1990. This plasmid contains the NDV-HN sequences linked to the 3'end of the vaccinia virus H6 promoter in a pUC9 vector. A HindIII -EcoRV fragment encompassing the 5' end of the vaccinia virus H6 promoterwas inserted into the HindIII and EcoRV sites of pRW802 to form pRW830.A perfect 3' end for NDV-HN was obtained by inserting the annealed andkinased oligonucleotides CE162 (SEQ ID NO:58) and CE163 (SEQ ID NO:59)into the EcoRI site of pRW830 to form pCE59, the final cassette forNDV-HN.

    CE162:                                                                            AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATTTAATTTTTATAAGCTTG       CE163:                                                                            AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTAGTAAAGGAACGATCCTG   

Construction of FPV Insertion Vector.

Plasmid pRW731-15 contains a 10 kb PvuII - PvuII fragment cloned fromgenomic DNA. The nucleotide sequence was determined on both strands fora 3660 bp PvuII - EcoRV fragment. The limits of an open reading framedesignated here as F8 were determined. Plasmid pRW761 is a sub-clone ofpRW731-15 containing a 2430 bp EcoRV - EcoRV fragment. The F8 ORF wasentirely contained between an XbaI site and an SspI site in pRW761. Inorder to create an insertion plasmid which on recombination with TROVACgenomic DNA would eliminate the F8 ORF, the following steps werefollowed. Plasmid pRW761 was completely digested with XbaI and partiallydigested with SspI. A 3700 bp XbaI - SspI band was isolated from the geland ligated with the annealed double-stranded oligonucleotides JCA017(SEQ ID NO:60) and JCA018 (SEQ ID NO:61).

    JCA017:5' CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGGGATCCTTATACGGGGAA    TAAT                                                                          JCA018:5' ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATATTAAAAAACATAAAGTG

The plasmid resulting from this ligation was designated pJCA002.

Construction of Double Insertion Vector for NDV F and Hn.

The H6 promoted NDV-HN sequence was inserted into the H6 promoted NDV-Fcassette by cloning a HindIII fragment from pCE59 that had been filledin with Klenow fragment of E. coli DNA polymerase into the HpaI site ofpCE71 to form pCE80. Plasmid pCE80 was completely digested with NdeI andpartially digested with BglII to generate an NdeI - BglII 4760 bpfragment containing the NDV F and HN genes both driven by the H6promoter and linked to F8 flanking arms. Plasmid pJCA021 was obtained byinserting a 4900 bp PvuII - HindII fragment from pRW731-15 into the SmaIand HindII sites of pBSSK+. Plasmid pJCA021 was then digested with NdeIand BglII and ligated to the 4760 bp NdeI - BglII fragment of pCE80 toform pJCA024. Plasmid pJCA024 therefore contains the NDV-F and HN genesinserted in opposite orientation with 3' ends adjacent between FPVflanking arms. Both genes are linked to the vaccinia virus H6 promoter.The right flanking arm adjacent to the NDV-F sequence consists of 2350bp of FPV sequence. The left flanking arm adjacent to the NDV-HNsequence consists of 1700 bp of FPV sequence.

Development of TROVAC-NDV.

Plasmid pJCA024 was transfected into TROVAC infected primary CEF cellsby using the calcium phosphate precipitation method previously described(Panicali et al., 1982; Piccini et al., 1987). Positive plaques wereselected on the basis of hybridization to specific NDV-F and HNradiolabelled probes and subjected to five sequential rounds of plaquepurification until a pure population was achieved. One representativeplaque was then amplified and the resulting TROVAC recombinant wasdesignated TROVAC-NDV (vFP96).

Immunofluorescence.

Indirect immunofluorescence was performed as described (Taylor et al.,1990) using a polyclonal anti-NDV serum and, as mono-specific reagents,sera produced in rabbits against vaccinia virus recombinants expressingNDV-F or NDV-HN.

Immunoprecipitation.

Immunoprecipitation reactions were performed as described (Taylor etal., 1990) using a polyclonal anti-NDV serum obtained from SPAFAS Inc.,Storrs, Conn.

The stock virus was screened by in situ plaque hybridization to confirmthat the F8 ORF was deleted. The correct insertion of the NDV genes intothe TROVAC genome and the deletion of the F8 ORF was also confirmed bySouthern blot hybridization.

In NDV-infected cells, the F glycoprotein is anchored in the membranevia a hydrophobic transmembrane region near the carboxyl terminus andrequires post-translational cleavage of a precursor, F₀, into twodisulfide linked polypeptides F₁ and F₂. Cleavage of F₀ is important indetermining the pathogenicity of a given NDV strain (Homma and Ohuchi,1973; Nagai et al., 1976; Nagai et al., 1980), and the sequence of aminoacids at the cleavage site is therefore critical in determining viralvirulence. It has been determined that amino acids at the cleavage sitein the NDV-F sequence inserted into FPV to form recombinant vFP29 hadthe sequence Arg - Arg - Gln - Arg - Arg (SEQ ID NO:42) (Taylor et al.,1990) which conforms to the sequence found to be a requirement forvirulent NDV strains (Chambers et al., 1986; Espion et al., 1987; Le etal., 1988; McGinnes and Morrison, 1986; Toyoda et al., 1987). The HNglycoprotein synthesized in cells infected with virulent strains of NDVis an uncleaved glycoprotein of 74 kDa. Extremely avirulent strains suchas Ulster and Queensland encode an HN precursor (HNo) which requirescleavage for activation (Garten et al., 1980).

The expression of F and HN genes in TROVAC-NDV was analyzed to confirmthat the gene products were authentically processed and presented.Indirect-immunofluorescence using a polyclonal anti-NDV chicken serumconfirmed that immunoreactive proteins were presented on the infectedcell surface. To determine that both proteins were presented on theplasma membrane, mono-specific rabbit sera were produced againstvaccinia recombinants expressing either the F or HN glycoproteins.Indirect immunofluorescence using these sera confirmed the surfacepresentation of both proteins.

Immunoprecipitation experiments were performed by using (³⁵ S)methionine labeled lysates of CEF cells infected with parental andrecombinant viruses. The expected values of apparent molecular weightsof the glycosylated forms of F₁ and F₂ are 54.7 and 10.3 kDarespectively (Chambers et al., 1986). In the immunoprecipitationexperiments using a polyclonal anti-NDV serum, fusion specific productsof the appropriate size were detected from the NDV-F single recombinantvFP29 (Taylor et al., 1990) and the TROVAC-NDV double recombinant vFP96.The HN glycoprotein of appropriate size was also detected from theNDV-HN single recombinant VFP-47 (Edbauer et al., 1990) and TROVAC-NDV.No NDV specific products were detected from uninfected and parentalTROVAC infected CEF cells.

In CEF cells, the F and HN glycoproteins are appropriately presented onthe infected cell surface where they are recognized by NDV immune serum.Immunoprecipitation analysis indicated that the F₀ protein isauthentically cleaved to the F₁ and F₂ components required in virulentstrains. Similarly, the HN glycoprotein was authentically processed inCEF cells infected with recombinant TROVAC-NDV.

Previous reports (Taylor et al., 1990; Edbauer et al., 1990; Boursnellet al., 1990a,b,c; Ogawa et al., 1990) would indicate that expression ofeither HN or F alone is sufficient to elicit protective immunity againstNDV challenge. Work on other paramyxoviruses has indicated, however,that antibody to both proteins may be required for full protectiveimmunity. It has been demonstrated that SV5 virus could spread in tissueculture in the presence of antibody to the HN glycoprotein but not tothe F glycoprotein (Merz et al., 1980). In addition, it has beensuggested that vaccine failures with killed measles virus vaccines weredue to inactivation of the fusion component (Norrby et al., 1975). Sinceboth NDV glycoproteins have been shown to be responsible for elicitingvirus neutralizing antibody (Avery et al., 1979) and both glycoproteins,when expressed individually in a fowlpox vector are able to induce aprotective immune response, it can be appreciated that the mostefficacious NDV vaccine should express both glycoproteins.

Example 21 Construction of ALVAC Recombinants Expressing Rabies VirusGlycoprotein G

This example describes the development of ALVAC, a canarypox virusvector and, of a canarypox-rabies recombinant designated as ALVAC-RG(vCP65) and its safety and efficacy.

Cells and Viruses.

The parental canarypox virus (Rentschler strain) is a vaccinal strainfor canaries. The vaccine strain was obtained from a wild type isolateand attenuated through more than 200 serial passages on chick embryofibroblasts. A master viral seed was subjected to four successive plaquepurifications under agar and one plaque clone was amplified through fiveadditional passages after which the stock virus was used as the parentalvirus in in vitro recombination tests. The plaque purified canarypoxisolate is designated ALVAC.

Construction of a Canarypox Insertion Vector.

An 880 bp canarypox PvuII fragment was cloned between the PvuII sites ofpUC9 to form pRW764.5. The sequence of this fragment is shown in FIG. 17(SEQ ID NO:62) between positions 1372 and 2251. The limits of an openreading frame designated as C5 were defined. It was determined that theopen reading frame was initiated at position 166 within the fragment andterminated at position 487. The C5 deletion was made withoutinterruption of open reading frames. Bases from position 167 throughposition 455 were replaced with the sequence (SEQ ID NO:63)GCTTCCCGGGAATTCTAGCTAGCTAGTTT. This replacement sequence containsHindIII, SmaI and EcoRI insertion sites followed by translation stopsand a transcription termination signal recognized by vaccinia virus RNApolymerase (Yuen et al., 1987). Deletion of the C5 ORF was performed asdescribed below. Plasmid pRW764.5 was partially cut with RsaI and thelinear product was isolated. The RsaI linear fragment was recut withBglII and the pRW764.5 fragment now with a RsaI to BglII deletion fromposition 156 to position 462 was isolated and used as a vector for thefollowing synthetic oligonucleotides:

RW145 (SEQ ID NO:64):

ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA

RW146 (SEQ ID NO:65):

GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT

Oligonucleotides RW145 and RW146 were annealed and inserted into the pRW764.5 RsaI and BglII vector described above. The resulting plasmid isdesignated pRW831.

Construction of Insertion Vector Containing the Rabies G Gene.

Construction of pRW838 is illustrated below. Oligonucleotides A throughE, which overlap the translation initiation codon of the H6 promoterwith the ATG of rabies G, were cloned into pUC9 as pRW737.Oligonucleotides A through E contain the H6 promoter, starting at NruI,through the HindIII site of rabies G followed by BglII. Sequences ofoligonucleotides A through E ((SEQ ID NO:66)-(SEQ ID NO:70)) are:

    A (SEQ ID NO:66)                                                                         CTGAAATTATTTCATTATCGCGATATCCGTTAA                                             GTTTGTATCGTAATGGTTCCTCAGGCTCTCCTGTTTGT                             B (SEQ ID NO:67)                                                                         CATTACGATACAAACTTAACGGATATCGCGATAA TGAAATAATTTCAG                  C (SEQ ID NO:68)                                                                         ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAA                                            TTCCCTATTTACACGATCCCAGACAAGCTTAGATCTCAG                            D (SEQ ID NO:69)                                                                         CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATA GGGAATTTCCCAAAACA               E (SEQ ID NO:70)                                                                         CAACGGAAAAACCAGAAGGGGTACAAACAGGACA GCCTGAGGAAC                 

The diagram of annealed oligonucleotides A through E is as follows:

               A                            C                                     ----------------------------------------------------.linevert split.------    ----------------------------------------------                                ----------------------------------.linevert split.------------------------    --------------.linevert split.------------------------------                       B                     E                D                             

Oligonucleotides A through E were kinased, annealed (95° C. for 5minutes, then cooled to room temperature), and inserted between thePvuII sites of pUC9. The resulting plasmid, pRW737, was cut with HindIIIand BglII and used as a vector for the 1.6 kbp HindIII-BglII fragment ofptg155PRO (Kieny et al., 1984) generating pRW739. The ptg155PRO HindIIIsite is 86 bp downstream of the rabies G translation initiation codon.BglII is downstream of the rabies G translation stop codon in ptg155PRO.pRW739 was partially cut with NruI, completely cut with BglII, and a 1.7kbp NruI-BglII fragment, containing the 3' end of the H6 promoterpreviously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkuset al., 1989) through the entire rabies G gene, was inserted between theNruI and BamHI sites of pRW824. The resulting plasmid is designatedpRW832. Insertion into pRW824 added the H6 promoter 5' of NruI. ThepRW824 sequence of BamHI followed by SmaI is (SEQ ID NO:71):GGATCCCCGGG. pRW824 is a plasmid that contains a nonpertinent genelinked precisely to the vaccinia virus H6 promoter. Digestion with NruIand BamHI completely excised this nonpertinent gene. The 1.8 kbp pRW832SmaI fragment, containing H6 promoted rabies G, was inserted into theSmaI of pRW831, to form plasmid pRW838.

Development of ALVAC-RG.

Plasmid pRW838 was transfected into ALVAC infected primary CEF cells byusing the calcium phosphate precipitation method previously described(Panicali et al., 1982; Piccini et al., 1987). Positive plaques wereselected on the basis of hybridization to a specific rabies G probe andsubjected to 6 sequential rounds of plaque purification until a purepopulation was achieved. One representative plaque was then amplifiedand the resulting ALVAC recombinant was designated ALVAC-RG (vCP65) (seealso FIGS. 18A and 18B). The correct insertion of the rabies G gene intothe ALVAC genome without subsequent mutation was confirmed by sequenceanalysis.

Immunofluorescence.

During the final stages of assembly of mature rabies virus particles,the glycoprotein component is transported from the golgi apparatus tothe plasma membrane where it accumulates with the carboxy terminusextending into the cytoplasm and the bulk of the protein on the externalsurface of the cell membrane. In order to confirm that the rabiesglycoprotein expressed in ALVAC-RG was correctly presented,immunofluorescence was performed on primary CEF cells infected withALVAC or ALVAC-RG. Immunofluorescence was performed as previouslydescribed (Taylor et al., 1990) using a rabies G monoclonal antibody.Strong surface fluorescence was detected on CEF cells infected withALVAC-RG but not with the parental ALVAC.

Immunoprecipitation.

Preformed monolayers of primary CEF, Vero (a line of African Greenmonkey kidney cells ATCC # CCL81) and MRC-5 cells (a fibroblast-likecell line derived from normal human fetal lung tissue ATCC # CCL171)were inoculated at 10 pfu per cell with parental virus ALVAC andrecombinant virus ALVAC-RG in the presence of radiolabelled ³⁵S-methionine and treated as previously described (Taylor et al., 1990).Immunoprecipitation reactions were performed using a rabies G specificmonoclonal antibody. Efficient expression of a rabies specificglycoprotein with a molecular weight of approximately 67 kDa wasdetected with the recombinant ALVAC-RG. No rabies specific products weredetected in uninfected cells or cells infected with the parental ALVACvirus.

Sequential Passaging Experiment.

In studies with ALVAC virus in a range of non-avian species noproliferative infection or overt disease was observed (Taylor et al.,1991b). However, in order to establish that neither the parental norrecombinant virus could be adapted to grow in non-avian cells, asequential passaging experiment was performed.

The two viruses, ALVAC and ALVAC-RG, were inoculated in 10 sequentialblind passages in three cell lines:

(1) Primary chick embryo fibroblast (CEF) cells produced from 11 day oldwhite leghorn embryos;

(2) Vero cells--a continuous line of African Green monkey kidney cells(ATCC # CCL81); and

(3) MRC-5 cells--a diploid cell line derived from human fetal lungtissue (ATCC # CCL171).

The initial inoculation was performed at an m.o.i. of 0.1 pfu per cellusing three 60 mm dishes of each cell line containing 2×10⁶ cells perdish. One dish was inoculated in the presence of 40 μg/ml of Cytosinearabinoside (Ara C), an inhibitor of DNA replication. After anabsorption period of 1 hour at 37° C., the inoculum was removed and themonolayer washed to remove unabsorbed virus. At this time the medium wasreplaced with 5 ml of EMEM+2% NBCS on two dishes (samples t0 and t7) and5 ml of EMEM+2% NBCS containing 40 μg/ml Ara C on the third (samplet7A). Sample t0 was frozen at -70° C. to provide an indication of theresidual input virus. Samples t7 and t7A were incubated at 37° C. for 7days, after which time the contents were harvested and the cellsdisrupted by indirect sonication.

One ml of sample t7 of each cell line was inoculated undiluted ontothree dishes of the same cell line (to provide samples t0, t7 and t7A)and onto one dish of primary CEF cells. Samples t0, t7 and t7A weretreated as for passage one. The additional inoculation on CEF cells wasincluded to provide an amplification step for more sensitive detectionof virus which might be present in the non-avian cells.

This procedure was repeated for 10 (CEF and MRC-5) or 8 (Vero)sequential blind passages. Samples were then frozen and thawed threetimes and assayed by titration on primary CEF monolayers.

Virus yield in each sample was then determined by plaque titration onCEF monolayers under agarose. Summarized results of the experiment areshown in Tables 5 and 6.

The results indicate that both the parental ALVAC and the recombinantALVAC-RG are capable of sustained replication on CEF monolayers with noloss of titer. In Vero cells, levels of virus fell below the level ofdetection after 2 passages for ALVAC and 1 passage for ALVAC-RG. InMRC-5 cells, a similar result was evident, and no virus was detectedafter 1 passage. Although the results for only four passages are shownin Tables 5 and 6 the series was continued for 8 (Vero) and 10 (MRC-5)passages with no detectable adaptation of either virus to growth in thenon-avian cells.

In passage 1 relatively high levels of virus were present in the t7sample in MRC-5 and Vero cells. However this level of virus wasequivalent to that seen in the t0 sample and the t7A sample incubated inthe presence of Cytosine arabinoside in which no viral replication canoccur. This demonstrated that the levels of virus seen at 7 days innon-avian cells represented residual virus and not newly replicatedvirus.

In order to make the assay more sensitive, a portion of the 7 dayharvest from each cell line was inoculated onto a permissive CEFmonolayer and harvested at cytopathic effect (CPE) or at 7 days if noCPE was evident. The results of this experiment are shown in Table 7.Even after amplification through a permissive cell line, virus was onlydetected in MRC-5 and Vero cells for two additional passages. Theseresults indicated that under the conditions used, there was noadaptation of either virus to growth in Vero or MRC-5 cells.

Inoculation of Macaques.

Four HIV seropositive macaques were initially inoculated with ALVAC-RGas described in Table 8. After 100 days these animals were re-inoculatedto determine a booster effect, and an additional seven animals wereinoculated with a range of doses. Blood was drawn at appropriateintervals and sera analyzed, after heat inactivation at 56° C. for 30minutes, for the presence of anti-rabies antibody using the RapidFluorescent Focus Inhibition Assay (Smith et al., 1973).

Inoculation of Chimpanzees.

Two adult male chimpanzees (50 to 65 kg weight range) were inoculatedintramuscularly or subcutaneously with 1×10⁷ pfu of vCP65. Animals weremonitored for reactions and bled at regular intervals for analysis forthe presence of anti-rabies antibody with the RFFI test (Smith et al.,1973). Animals were re-inoculated with an equivalent dose 13 weeks afterthe initial inoculation.

Inoculation of Mice.

Groups of mice were inoculated with 50 to 100 μl of a range of dilutionsof different batches of vCP65. Mice were inoculated in the footpad. Onday 14, mice were challenged by intracranial inoculation of from 15 to43 mouse LD₅₀ of the virulent CVS strain of rabies virus. Survival ofmice was monitored and a protective dose 50% (PD₅₀) calculated at 28days post-inoculation.

Inoculation of Dogs and Cats.

Ten beagle dogs, 5 months old, and 10 cats, 4 months old, wereinoculated subcutaneously with either 6.7 or 7.7 log₁₀ TCID₅₀ ofALVAC-RG. Four dogs and four cats were not inoculated. Animals were bledat 14 and 28 days post-inoculation and anti-rabies antibody assessed inan RFFI test. The animals receiving 6.7 log₁₀ TCID₅₀ of ALVAC-RG werechallenged at 29 days post-vaccination with 3.7 log₁₀ mouse LD₅₀ (dogs)or 4.3 log₁₀ mouse LD₅₀ (cats) of the NYGS rabies virus challengestrain.

Inoculation of Squirrel Monkeys.

Three groups of four squirrel monkeys (Saimiri sciureus) were inoculatedwith one of three viruses (a) ALVAC, the parental canarypox virus, (b)ALVAC-RG, the recombinant expressing the rabies G glycoprotein or (c)vCP37, a canarypox recombinant expressing the envelope glycoprotein offeline leukemia virus. Inoculations were performed under ketamineanaesthesia. Each animal received at the same time: (1) 20 μl instilledon the surface of the right eye without scarification; (2) 100 μl asseveral droplets in the mouth; (3) 100 μl in each of two intradermalinjection sites in the shaven skin of the external face of the rightarm; and (4) 100 μl in the anterior muscle of the right thigh.

Four monkeys were inoculated with each virus, two with a total of 5.0log₁₀ pfu and two with a total of 7.0 log₁₀ pfu. Animals were bled atregular intervals and sera analyzed for the presence of antirabiesantibody using an RFFI test (Smith et al., 1973). Animals were monitoreddaily for reactions to vaccination. Six months after the initialinoculation the four monkeys receiving ALVAC-RG, two monkeys initiallyreceiving vCP37, and two monkeys initially receiving ALVAC, as well asone naive monkey were inoculated with 6.5 log₁₀ pfu of ALVAC-RGsubcutaneously. Sera were monitored for the presence of rabiesneutralizing antibody in an RFFI test (Smith et al., 1973).

Inoculation of Human Cell Lines with ALVAC-RG.

In order to determine whether efficient expression of a foreign genecould be obtained in non-avian cells in which the virus does notproductively replicate, five cell types, one avian and four non-avian,were analyzed for virus yield, expression of the foreign rabies G geneand viral specific DNA accumulation. The cells inoculated were:

(a) Vero, African Green monkey kidney cells, ATCC # CCL81;

(b) MRC-5, human embryonic lung, ATCC # CCL 171;

(c) WISH human amnion, ATCC # CCL 25;

(d) Detroit-532, human foreskin, Downs's syndrome, ATCC # CCL 54; and

(e) Primary CEF cells.

Chicken embryo fibroblast cells produced from 11 day old white leghornembryos were included as a positive control. All inoculations wereperformed on preformed monolayers of 2×10⁶ cells as discussed below.

A. Methods for DNA Analysis.

Three dishes of each cell line were inoculated at 5 pfu/cell of thevirus under test, allowing one extra dish of each cell lineun-inoculated. One dish was incubated in the presence of 40 μg/ml ofcytosine arabinoside (Ara C). After an adsorption period of 60 minutesat 37° C., the inoculum was removed and the monolayer washed twice toremove unadsorbed virus. Medium (with or without Ara C) was thenreplaced. Cells from one dish (without Ara C) were harvested as a timezero sample. The remaining dishes were incubated at 37° C. for 72 hours,at which time the cells were harvested and used to analyze DNAaccumulation. Each sample of 2×10⁶ cells was resuspended in 0.5 mlphosphate buffered saline (PBS) containing 40 mM EDTA and incubated for5 minutes at 37° C. An equal volume of 1.5% agarose prewarmed at 42° C.and containing 120 mM EDTA was added to the cell suspension and gentlymixed. The suspension was transferred to an agarose plug mold andallowed to harden for at least 15 min. The agarose plugs were thenremoved and incubated for 12-16 hours at 50° C. in a volume of lysisbuffer (1% sarkosyl, 100 μg/ml proteinase K, 10 mM Tris HCl pH 7.5, 200mM EDTA) that completely covers the plug. The lysis buffer was thenreplaced with 5.0 ml sterile 0.5×TBE (44.5 mM Tris-borate, 44.5 mM boricacid, 0.5 mM EDTA) and equilibrated at 4° C. for 6 hours with 3 changesof TBE buffer. The viral DNA within the plug was fractionated fromcellular RNA and DNA using a pulse field electrophoresis system.Electrophoresis was performed for 20 hours at 180 V with a ramp of 50-90sec at 15° C. in 0.5×TBE. The DNA was run with lambda DNA molecularweight standards. After electrophoresis the viral DNA band wasvisualized by staining with ethidium bromide. The DNA was thentransferred to a nitrocellulose membrane and probed with a radiolabelledprobe prepared from purified ALVAC genomic DNA.

B. Estimation of Virus Yield.

Dishes were inoculated exactly as described above, with the exceptionthat input multiplicity was 0.1 pfu/cell. At 72 hours post infection,cells were lysed by three successive cycles of freezing and thawing.Virus yield was assessed by plaque titration on CEF monolayers.

C. Analysis of Expression of Rabies G Gene.

Dishes were inoculated with recombinant or parental virus at amultiplicity of 10 pfu/cell, allowing an additional dish as anuninfected virus control. After a one hour absorption period, the mediumwas removed and replaced with methionine free medium. After a 30 minuteperiod, this medium was replaced with methionine-free medium containing25 uCi/ml of ³⁵ S-Methionine. Infected cells were labelled overnight(approximately 16 hours), then lysed by the addition of buffer A lysisbuffer. Immunoprecipitation was performed as previously described(Taylor et al., 1990) using a rabies G specific monoclonal antibody.

Results: Estimation of Viral Yield.

The results of titration for yield at 72 hours after inoculation at 0.1pfu per cell are shown in Table 9. The results indicate that while aproductive infection can be attained in the avian cells, no increase invirus yield can be detected by this method in the four non-avian cellsystems.

Analysis of Viral DNA Accumulation.

In order to determine whether the block to productive viral replicationin the non-avian cells occurred before or after DNA replication, DNAfrom the cell lysates was fractionated by electrophoresis, transferredto nitrocellulose and probed for the presence of viral specific DNA. DNAfrom uninfected CEF cells, ALVAC-RG infected CEF cells at time zero,ALVAC-RG infected CEF cells at 72 hours post-infection and ALVAC-RGinfected CEF cells at 72 hours post-infection in the presence of 40μg/ml of cytosine arabinoside all showed some background activity,probably due to contaminating CEF cellular DNA in the radiolabelledALVAC DNA probe preparation. However, ALVAC-RG infected CEF cells at 72hours post-infection exhibited a strong band in the region ofapproximately 350 kbp representing ALVAC-specific viral DNAaccumulation. No such band is detectable when the culture is incubatedin the presence of the DNA synthesis inhibitor, cytosine arabinoside.Equivalent samples produced in Vero cells showed a very faint band atapproximately 350 kbp in the ALVAC-RG infected Vero cells at time zero.This level represented residual virus. The intensity of the band wasamplified at 72 hours post-infection indicating that some level of viralspecific DNA replication had occurred in Vero cells which had notresulted in an increase in viral progeny. Equivalent samples produced inMRC-5 cells indicated that no viral specific DNA accumulation wasdetected under these conditions in this cell line. This experiment wasthen extended to include additional human cell lines, specifically WISHand Detroit-532 cells. ALVAC infected CEF cells served as a positivecontrol. No viral specific DNA accumulation was detected in either WISHor Detroit cells inoculated with ALVAC-RG. It should be noted that thelimits of detection of this method have not been fully ascertained andviral DNA accumulation may be occurring, but at a level below thesensitivity of the method. Other experiments in which viral DNAreplication was measured by ³ H-thymidine incorporation support theresults obtained with Vero and MRC-5 cells.

Analysis of Rabies Gene Expression.

To determine if any viral gene expression, particularly that of theinserted foreign gene, was occurring in the human cell lines even in theabsence of viral DNA replication, immunoprecipitation experiments wereperformed on ³⁵ S-methionine labelled lysates of avian and non-aviancells infected with ALVAC and ALVAC-RG. The results ofimmunoprecipitation using a rabies G specific monoclonal antibodyillustrated specific immunoprecipitation of a 67 kDa glycoprotein inCEF, Vero and MRC-5, WISH and Detroit cells infected with ALVAC-RG. Nosuch specific rabies gene products were detected in any of theuninfected and parentally infected cell lysates.

The results of this experiment indicated that in the human cell linesanalyzed, although the ALVAC-RG recombinant was able to initiate aninfection and express a foreign gene product under the transcriptionalcontrol of the H6 early/late vaccinia virus promoter, the replicationdid not proceed through DNA replication, nor was there any detectableviral progeny produced. In the Vero cells, although some level ofALVAC-RG specific DNA accumulation was observed, no viral progeny wasdetected by these methods. These results would indicate that in thehuman cell lines analyzed the block to viral replication occurs prior tothe onset of DNA replication, while in Vero cells, the block occursfollowing the onset of viral DNA replication.

In order to determine whether the rabies glycoprotein expressed inALVAC-RG was immunogenic, a number of animal species were tested byinoculation of the recombinant. The efficacy of current rabies vaccinesis evaluated in a mouse model system. A similar test was thereforeperformed using ALVAC-RG. Nine different preparations of virus(including one vaccine batch (J) produced after 10 serial tissue culturepassages of the seed virus) with infectious titers ranging from 6.7 to8.4 log₁₀ TCID₅₀ per ml were serially diluted and 50 to 100 μl ofdilutions inoculated into the footpad of four to six week old mice. Micewere challenged 14 days later by the intracranial route with 300 μl ofthe CVS strain of rabies virus containing from 15 to 43 mouse LD₅₀ asdetermined by lethality titration in a control group of mice. Potency,expressed as the PD₅₀ (Protective dose 50%), was calculated at 14 dayspost-challenge. The results of the experiment are shown in Table 10. Theresults indicated that ALVAC-RG was consistently able to protect miceagainst rabies virus challenge with a PD₅₀ value ranging from 3.33 to4.56 with a mean value of 3.73 (STD 0.48). As an extension of thisstudy, male mice were inoculated intracranially with 50 μl of viruscontaining 6.0 log₁₀ TCID₅₀ of ALVAC-RG or with an equivalent volume ofan uninfected cell suspension. Mice were sacrificed on days 1, 3 and 6post-inoculation and their brains removed, fixed and sectioned.Histopathological examination showed no evidence for neurovirulence ofALVAC-RG in mice.

In order to evaluate the safety and efficacy of ALVAC-RG for dogs andcats, a group of 14, 5 month old beagles and 14, 4 month old cats wereanalyzed. Four animals in each species were not vaccinated. Five animalsreceived 6.7 log₁₀ TCID₅₀ subcutaneously and five animals received 7.7log₁₀ TCID₅₀ by the same route. Animals were bled for analysis foranti-rabies antibody. Animals receiving no inoculation or 6.7 log₁₀TCID₅₀ of ALVAC-RG were challenged at 29 days post-vaccination with 3.7log₁₀ mouse LD₅₀ (dogs, in the temporal muscle) or 4.3 log₁₀ mouse LD₅₀(cats, in the neck) of the NYGS rabies virus challenge strain. Theresults of the experiment are shown in Table 11.

No adverse reactions to inoculation were seen in either cats or dogswith either dose of inoculum virus. Four of 5 dogs immunized with 6.7log₁₀ TCID₅₀ had antibody titers on day 14 post-vaccination and all dogshad titers at 29 days. All dogs were protected from a challenge whichkilled three out of four controls. In cats, three of five cats receiving6.7 log₁₀ TCID₅₀ had specific antibody titers on day 14 and all catswere positive on day 29 although the mean antibody titer was low at 2.9IU. Three of five cats survived a challenge which killed all controls.All cats immunized with 7.7 log₁₀ TCID₅₀ had antibody titers on day 14and at day 29 the Geometric Mean Titer was calculated as 8.1International Units.

The immune response of squirrel monkeys (Saimiri sciureus) toinoculation with ALVAC, ALVAC-RG and an unrelated canarypox virusrecombinant was examined. Groups of monkeys were inoculated as describedabove and sera analyzed for the presence of rabies specific antibody.Apart from minor typical skin reactions to inoculation by theintradermal route, no adverse reactivity was seen in any of the monkeys.Small amounts of residual virus were isolated from skin lesions afterintradermal inoculation on days two and four post-inoculation only. Allspecimens were negative on day seven and later. There was no localreaction to intra-muscular injection. All four monkeys inoculated withALVAC-RG developed anti-rabies serum neutralizing antibodies as measuredin an RFFI test. Approximately six months after the initial inoculationall monkeys and one additional naive monkey were re-inoculated by thesubcutaneous route on the external face of the left thigh with 6.5 log₁₀TCID₅₀ of ALVAC-RG. Sera were analyzed for the presence of anti-rabiesantibody. The results are shown in Table 12.

Four of the five monkeys naive to rabies developed a serologicalresponse by seven days post-inoculation with ALVAC-RG. All five monkeyshad detectable antibody by 11 days post-inoculation. Of the four monkeyswith previous exposure to the rabies glycoprotein, all showed asignificant increase in serum neutralization titer between days 3 and 7post-vaccination. The results indicate that vaccination of squirrelmonkeys with ALVAC-RG does not produce adverse side-effects and aprimary neutralizing antibody response can be induced. An amnanesticresponse is also induced on re-vaccination. Prior exposure to ALVAC orto a canarypox recombinant expressing an unrelated foreign gene does notinterfere with induction of an anti-rabies immune response uponre-vaccination.

The immunological response of HIV-2 seropositive macaques to inoculationwith ALVAC-RG was assessed. Animals were inoculated as described aboveand the presence of anti-rabies serum neutralizing antibody assessed inan RFFI test. The results, shown in Table 13, indicated that HIV-2positive animals inoculated by the subcutaneous route developedanti-rabies antibody by 11 days after one inoculation. An anamnesticresponse was detected after a booster inoculation given approximatelythree months after the first inoculation. No response was detected inanimals receiving the recombinant by the oral route. In addition, aseries of six animals were inoculated with decreasing doses of ALVAC-RGgiven by either the intra-muscular or subcutaneous routes. Five of thesix animals inoculated responded by 14 days post-vaccination with nosignificant difference in antibody titer.

Two chimpanzees with prior exposure to HIV were inoculated with 7.0log₁₀ pfu of ALVAC-RG by the subcutaneous or intra-muscular route. At 3months post-inoculations both animals were re-vaccinated in an identicalfashion. The results are shown in Table 14.

No adverse reactivity to inoculation was noted by either intramuscularor subcutaneous routes. Both chimpanzees responded to primaryinoculation by 14 days and a strongly rising response was detectedfollowing re-vaccination.

                  TABLE 5                                                         ______________________________________                                        Sequential Passage of ALVAC in Avian and non-Avian Cells.                               CEF        Vero   MRC-5                                             ______________________________________                                        Pass 1                                                                        Sample   to.sup.a                                                                             2.4          3.0  2.6                                                  t7.sup.b                                                                             7.0          1.4  0.4                                                  t7A.sup.c                                                                            1.2          1.2  0.4                                         Pass 2                                                                        Sample   to     5.0          0.4   N.D..sup.d                                          t7     7.3          0.4  N.D.                                                 t7A    3.9          N.D. N.D.                                        Pass 3                                                                        Sample   to     5.4          0.4  N.D.                                                 t7     7.4          N.D. N.D.                                                 t7A    3.8          N.D. N.D.                                        Pass 4                                                                        Sample   to     5.2          N.D. N.D.                                                 t7     7.1          N.D. N.D.                                                 t7A    3.9          N.D. N.D.                                        ______________________________________                                         .sup.a This sample was harvested at zero time and represents the residual     input virus. The titer is expressed as log.sub.10  pfu per ml.                .sup.b This sample was harvested at 7 days postinfection.                     .sup.c This sample was inoculated in the presence of 40 μg/ml of           Cytosine arabinoside and harvested at 7 days post infection.                  .sup.d Not detectable                                                    

                  TABLE 6                                                         ______________________________________                                        Sequential Passage of ALVAC-RG in Avian and non-Avian Cells                             CEF       Vero    MRC-5                                             ______________________________________                                        Pass 1                                                                        Sample   t0.sup.a                                                                             3.0         2.9   2.9                                                  t7.sup.b                                                                             7.1         1.0   1.4                                                  t7A.sup.c                                                                            1.8         1.4   1.2                                         Pass 2                                                                        Sample   t0     5.1         0.4   0.4                                                  t7     7.1          N.D..sup.d                                                                         N.D.                                                 t7A    3.8         N.D.  N.D.                                        Pass 3                                                                        Sample   t0     5.1         0.4   N.D.                                                 t7     7.2         N.D.  N.D.                                                 t7A    3.6         N.D.  N.D.                                        Pass 4                                                                        Sample   t0     5.1         N.D.  N.D.                                                 t7     7.0         N.D.  N.D.                                                 t7A    4.0         N.D.  N.D                                         ______________________________________                                         .sup.a This sample was harvested at zero time and represents the residual     input virus. The titer is expressed as log.sub.10  pfu per ml.                .sup.b This sample was harvested at 7 days postinfection.                     .sup.c This sample was inoculated in the presence of 40 μg/ml of           Cytosine arabinoside and harvested at 7 days postinfection.                   .sup.d Not detectable.                                                   

                  TABLE 7                                                         ______________________________________                                        Amplification of residual virus by passage in CEF cells                                CEF        Vero    MRC-5                                             ______________________________________                                        a) ALVAC                                                                      Pass     2.sup.a                                                                              7.0.sup.b   6.0   5.2                                                  3     7.5          4.1   4.9                                                  4     7.5          N.D..sup.c                                                                          N.D.                                                 5     7.1          N.D.  N.D.                                        b) ALVAC-RG                                                                   Pass     2a    7.2          5.5   5.5                                                  3     7.2          5.0   5.1                                                  4     7.2          N.D.  N.D.                                                 5     7.2          N.D.  N.D.                                        ______________________________________                                         .sup.a Pass 2 represents the amplification in CEF cells of the 7 day          sample from Pass 1.                                                           .sup.b Titer expressed as log.sub.10  pfu per ml                              .sup.c Not Detectable                                                    

                  TABLE 8                                                         ______________________________________                                        Schedule of inoculation of rhesus macaques with ALVAC-RG (vCP65)              Animal            Inoculation                                                 ______________________________________                                        176L   Primary:   1 × 10.sup.8  pfu of vCP65 orally in TANG                    Secondary: 1 × 10.sup.7  pfu of vCP65 plus 1 ×                               10.sup.7                                                                      pfu of VCP82.sup.a  by SC route                             185L   Primary:   1 × 10.sup.8  pfu of vCP65 orally in Tang                    Secondary: 1 × 10.sup.7  pfu of vCP65 plus 1 ×                               10.sup.7                                                                      pfu of vCP82 by SC route                                    177L   Primary:   5 × 10.sup.7  pfu SC of vCP65 by SC route                    Secondary: 1 × 10.sup.7  pfu of vCP65 plus 1 ×                               10.sup.7                                                                      pfu of vCP82 by SC route                                    186L   Primary:   5 × 10.sup.7  pfu of vCP65 by SC route                       Secondary: 1 × 10.sup.7  pfu of vCP65 plus 1 ×                               10.sup.7                                                                      pfu of vCP82 by SC route                                    178L   Primary:   1 × 10.sup.7  pfu of vCP65 by SC route                182L   Primary:   1 × 10.sup.7  pfu of vCP65 by IM route                179L   Primary:   1 × 10.sup.6  pfu of vCP65 by SC route                183L   Primary:   1 × 10.sup.6  pfu of vCP65 by IM route                180L   Primary:   1 × 10.sup.6  pfu of vCP65 by SC route                184L   Primary:   1 × 10.sup.5  pfu of vCP65 by IM route                187L   Primary    1 × 10.sup.7  pfu of vCP65 orally                     ______________________________________                                         .sup.a vCP82 is a canarypox virus recombinant expressing the measles viru     fusion and hemagglutinin genes.                                          

                  TABLE 9                                                         ______________________________________                                        Analysis of yield in avian and non-avian cells                                inoculated with ALVAC-RG                                                                Sample Time                                                         Cell Type   t0           t72    t72A.sup.b                                    ______________________________________                                        Expt 1                                                                        CEF          3.3.sup.a   7.4    1.7                                           Vero        3.0          1.4    1.7                                           MRC-5       3.4          2.0    1.7                                           Expt 2                                                                        CEF         2.9          7.5    <1.7                                          WISH        3.3          2.2    2.0                                           Detroit-532 2.8                                                                           1.7          <1.7                                                 ______________________________________                                         .sup.a Titer expressed as log.sub.10  pfu per ml                              .sup.b Culture incubated in the presence of 40 μg/ml of Cytosine           arabinoside                                                              

                  TABLE 10                                                        ______________________________________                                        Potency of ALVAC-RG as tested in mice                                         Test            Challenge Dose.sup.a                                                                      PD.sub.50 .sup.b                                  ______________________________________                                        Initial seed    43          4.56                                              Primary seed    23          3.34                                              Vaccine Batch H 23          4.52                                              Vaccine Batch I 23          3.33                                              Vaccine Batch K 15          3.64                                              Vaccine Batch L 15          4.03                                              Vaccine Batch M 15          3.32                                              Vaccine Batch N 15          3.39                                              Vaccine Batch J 23          3.42                                              ______________________________________                                         .sup.a Expressed as mouse LD.sub.50                                           .sup.b Expressed as log.sub.10  TCID.sub.50                              

                  TABLE 11                                                        ______________________________________                                        Efficacy of ALVAC-RG in dogs and cats                                                  Dogs            Cats                                                 Dose     Antibody.sup.a                                                                         Survival.sup.b                                                                           Antibody                                                                             Survival                                  ______________________________________                                        6.7      11.9     5/5        2.9    3/5                                       7.7      10.1     N.T.       8.1                                              N.T.                                                                          ______________________________________                                         .sup.a Antibody at day 29 post inoculation expressed as the geometric mea     titer in International Units.                                                 .sup.b Expressed as a ratio of survivors over animals challenged         

                  TABLE 12                                                        ______________________________________                                        Anti-rabies serological response of Squirrel monkeys                          inoculated with canarypox recombinants                                        Mon-                                                                          key  Previous   Rabies serum-neutralizing antibody.sup.a                      #    Exposure   -196.sup.b                                                                            0    3    7    11  21  28                             ______________________________________                                        22   ALVAC.sup.c                                                                              NT.sup.g                                                                              <1.2 <1.2 <1.2 2.1 2.3 2.2                            51   ALVAC.sup.c                                                                              NT      <1.2 <1.2 1.7  2.2 2.2 2.2                            39   vCP37.sup.d                                                                              NT      <1.2 <1.2 1.7  2.1 2.2  N.T..sup.g                    55   vCP37.sup.d                                                                              NT      <1.2 <1.2 1.7  2.2 2.1 N.T.                           37   ALVAC-RG.sup.e                                                                           2.2     <1.2 <1.2 3.2  3.5 3.5 3.2                            53   ALVAC-RG.sup.e                                                                           2.2     <1.2 <1.2 3.6  3.6 3.6 3.4                            38   ALVAC-RG.sup.f                                                                           2.7     <1.7 <1.7 3.2  3.8 3.6 N.T.                           54   ALVAC-RG.sup.f                                                                           3.2     <1.7 <1.5 3.6  4.2 4.0 3.6                            57   None       NT      <1.2 <1.2 1.7  2.7 2.7 2.3                            ______________________________________                                         .sup.a As determined by RFFI test on days indicated and expressed in          International Units                                                           .sup.b Day196 represents serum from day 28 after primary vaccination          .sup.c Animals received 5.0 log.sub.10  TCID.sub.50  of ALVAC                 .sup.d Animals received 5.0 log.sub.10  TCID.sub.50  of vCP37                 .sup.e Animals received 5.0 log.sub.10  TCID.sub.50  of ALVACRG               .sup.f Animals received 7.0 log.sub.10  TCID.sub.50  of ALVACRG               .sup.g Not tested.                                                       

                                      TABLE 13                                    __________________________________________________________________________    Inoculation of rhesus macaques with ALVAC-RG.sup.a                            Route of Primary Inoculation                                                  Days post-                                                                          or/Tang                                                                             SC SC SC IM SC IM SC IM OR                                        Inoculation                                                                         176L.sup.b 185L                                                                     177L                                                                             186L                                                                             178L                                                                             182L                                                                             179L                                                                             183L                                                                             180L                                                                             184L                                                                             187L.sup.b                                __________________________________________________________________________    -84   -- --    --                                                             -9    -- -- -- -- --    --                                                     3    -- -- -- --                                                              6    -- -- ±                                                                             ±                                                           11    -- --  16.sup.d                                                                        128                                                            19    -- --  32                                                                              128                                                                              --    --                                                    35    -- --  32                                                                              512                                                            59    -- --  64                                                                              256                                                            75    -- --  64                                                                              128                                                                              --    --                                                     99.sup.c                                                                           -- --  64                                                                              256                                                                              --    -- -- -- -- --                                         2    -- --  32                                                                              256                                                                              -- -- -- -- -- -- --                                         6    -- -- 512                                                                              512                                                                              -- -- -- -- -- -- --                                        15    16 16 512                                                                              512                                                                              64 32 64 128                                                                              32 -- --                                        29    16 32 256                                                                              256                                                                              64 64 32 128                                                                              32 -- --                                        55       32          32     32                                                                              16 --                                           57    16    128                                                                              128                                                                              16    16          --                                        __________________________________________________________________________     .sup.a See Table 9 for schedule of inoculations.                              .sup.b Animals 176L and 185L received 8.0 log.sub.10  pfu by the oral         route in 5 ml Tang. Animal 187L received 7.0 log.sub.10  pfu by oral rout     not in Tang.                                                                  .sup.c Day of revaccination for animals 176L, 185L, 177L and 186L by S.C.     route, and primary vaccination for animals 178L, 182L, 179L, 183L, 180L,      184L and 187L.                                                                .sup.d Titers expressed as reciprocal of last dilution showing inhibition     of fluorescence in an RFFI test.                                         

                  TABLE 14                                                        ______________________________________                                        Inoculation of chimpanzees with ALVAC-RG                                      Weeks post-    Animal 431                                                                              Animal 457                                           Inoculation    I.M.      S.C.                                                 ______________________________________                                        0               <8.sup.a         <8                                           1              <8                <8                                           2               8                32                                           4              16                32                                           8              16                32                                           12.sup.b /0    16                 8                                           13/1           128               128                                          15/3           256               512                                          20/8           64                128                                          26/12          32                128                                          ______________________________________                                         .sup.a Titer expressed as reciprocal of last dilution showing inhibition      of fluorescence in an RFFI test                                               .sup.b Day of reinoculation                                              

Example 22 Immunization of Humans Using Canarypox Expressing RabiesGlycoprotein (ALVAC-RG; vCP65)

ALVAC-RG (vCP65) was generated as described in Example 21 and FIGS. 18Aand 18B. For scaling-up and vaccine manufacturing ALVAC-RG (vCP65) wasgrown in primary CEF derived from specified pathogen free eggs. Cellswere infected at a multiplicity of 0.01 and incubated at 37° C. forthree days.

The vaccine virus suspension was obtained by ultrasonic disruption inserum free medium of the infected cells; cell debris were then removedby centrifugation and filtration. The resulting clarified suspension wassupplemented with lyophilization stabilizer (mixture of amino-acids),dispensed in single dose vials and freeze dried. Three batches ofdecreasing titer were prepared by ten-fold serial dilutions of the virussuspension in a mixture of serum free medium and lyophilizationstabilizer, prior to lyophilization.

Quality control tests were applied to the cell substrates, media andvirus seeds and final product with emphasis on the search foradventitious agents and innocuity in laboratory rodents. No undesirabletrait was found.

Preclinical Data.

Studies in vitro indicated that VERO or MRC-5 cells do not support thegrowth of ALVAC-RG (vCP65); a series of eight (VERO) and 10 (MRC) blindserial passages caused no detectable adaptation of the virus to grow inthese non avian lines. Analyses of human cell lines (MRC-5, WISH,Detroit 532, HEL, HNK or EBV-transformed lymphoblastoid cells) infectedor inoculated with ALVAC-RG (vCP65) showed no accumulation of virusspecific DNA suggesting that in these cells the block in replicationoccurs prior to DNA synthesis. Significantly, however, the expression ofthe rabies virus glycoprotein gene in all cell lines tested indicatingthat the abortive step in the canarypox replication cycle occurs priorto viral DNA replication.

The safety and efficacy of ALVAC-RG (vCP65) were documented in a seriesof experiments in animals. A number of species including canaries,chickens, ducks, geese, laboratory rodents (suckling and adult mice),hamsters, guinea-pigs, rabbits, cats and dogs, squirrel monkeys, rhesusmacaques and chimpanzees, were inoculated with doses ranging from 10⁵ to10⁸ pfu. A variety of routes were used, most commonly subcutaneous,intramuscular and intradermal but also oral (monkeys and mice) andintracerebral (mice).

In canaries, ALVAC-RG (vCP65) caused a "take" lesion at the site ofscarification with no indication of disease or death. Intradermalinoculation of rabbits resulted in a typical poxvirus inoculationreaction which did not spread and healed in seven to ten days. There wasno adverse side effects due to canarypox in any of the animal tests.Immunogenicity was documented by the development of anti-rabiesantibodies following inoculation of ALVAC-RG (vCP65) in rodents, dogs,cats, and primates, as measured by Rapid Fluorescent Focus InhibitionTest (RFFIT). Protection was also demonstrated by rabies virus challengeexperiments in mice, dogs, and cats immunized with ALVAC-RG (vCP65).

Volunteers.

Twenty-five healthy adults aged 20-45 with no previous history of rabiesimmunization were enrolled. Their health status was assessed by completemedical histories, physical examinations, hematological and bloodchemistry analyses. Exclusion criteria included pregnancy, allergies,immune depression of any kind, chronic debilitating disease, cancer,injection of immune globins in the past three months, and seropositivityto human immunodeficiency virus (HIV) or to hepatitis B virus surfaceantigen.

Study Design.

Participants were randomly allocated to receive either standard HumanDiploid Cell Rabies Vaccine (HDC) batch no E0751 (Pasteur Merieux Serums& Vaccine, Lyon, France) or the study vaccine ALVAC-RG (vCP65).

The trial was designated as a dose escalation study. Three batches ofexperimental ALVAC-RG (vCP65) vaccine were used sequentially in threegroups of volunteers (Groups A, B and C) with two week intervals betweeneach step. The concentration of the three batches was 10³.5, 10⁴.5,10⁵.5 Tissue Culture Infectious Dose (TCID₅₀) per dose, respectively.

Each volunteer received two doses of the same vaccine subcutaneously inthe deltoid region at an interval of four weeks. The nature of theinjected vaccine was not known by the participants at the time of thefirst injection but was known by the investigator.

In order to minimize the risk of immediate hypersensitivity at the timeof the second injection, the volunteers of Group B allocated to themedium dose of experimental vaccine were injected 1 h previously withthe lower dose and those allocated to the higher dose (Group C) receivedsuccessively the lower and the medium dose at hourly intervals.

Six months later, the recipients of the highest dosage of ALVAC-RG(vCP65) (Group C) and HDC vaccine were offered a third dose of vaccine;they were then randomized to receive either the same vaccine aspreviously or the alternate vaccine. As a result, four groups wereformed corresponding to the following immunization scheme: 1. HDC, HDC -HDC; 2. HDC, HDC - ALVAC-RG (vCP65); 3. ALVAC-RG (vCP65), ALVAC-RG(vCP65) - HDC; 4. ALVAC-RG (vCP65), ALVAC-RG (vCP65), ALVAC-RG (vCP65).

Monitoring of Side Effects.

All subjects were monitored for 1 h after injection and re-examinedevery day for the next five days. They were asked to record local andsystemic reactions for the next three weeks and were questioned bytelephone two times a week.

Laboratory Investigators.

Blood specimens were obtained before enrollment and two, four and sixdays after each injection. Analysis included complete blood cell count,liver enzymes and creatine kinase assays.

Antibody Assays.

Antibody assays were performed seven days prior to the first injectionand at days 7, 28, 35, 56, 173, 187 and 208 of the study.

The levels of neutralizing antibodies to rabies were determined usingthe Rapid Fluorescent Focus Inhibition test (RFFIT) (Smith & Yaeger, InLaboratory Techniques on Rabies). Canarypox antibodies were measured bydirect ELISA. The antigen, a suspension of purified canarypox virusdisrupted with 0.1% Triton X100, was coated in microplates. Fixeddilutions of the sera were reacted for two hours at room temperature andreacting antibodies were revealed with a peroxidase labelled anti-humanIgG goat serum. The results are expressed as the optical density read at490 nm.

Analysis.

Twenty-five subjects were enrolled and completed the study. There were10 males and 15 females and the mean age was 31.9 (21 to 48). All butthree subjects had evidence of previous smallpox vaccination; the threeremaining subjects had no typical scar and vaccination history. Threesubjects received each of the lower doses of experimental vaccine (10³.5and 10⁴.5 TCID₅₀), nine subjects received 10⁵.5 TCID₅₀ and ten receivedthe HDC vaccine.

Safety (Table 14).

During the primary series of immunization, fever greater than 37.7° C.was noted within 24 hours after injection in one HDC recipient (37.8°C.) and in one vCP65 10⁵.5 TCID₅₀ recipient (38° C.). No other systemicreaction attributable to vaccination was observed in any participant.

Local reactions were noted in 9/10 recipients of HDC vaccine injectedsubcutaneously and in 0/3, 1/3 and 9/9 recipients of vCP65 10³.5, 10⁴.5,10⁵.5 TCID₅₀, respectively.

Tenderness was the most common symptoms and was always mild. Other localsymptoms included redness and induration which were also mild andtransient. All symptoms usually subsided within 24 hours and neverlasted more than 72 hours.

There was no significant change in blood cell counts, liver enzymes orcreatine kinase values.

Immune Responses; Neutralizing Antibodies to Rabies (Table 16).

Twenty eight days after the first injection all the HDC recipients hadprotective titers (≧0.5 IU/ml). By contrast none in groups A and B(10³.5 and 10⁴.5 TCID₅₀) and only 2/9 in group C (10⁵.5 TCID₅₀) ALVAC-RG(vCP65) recipients reached this protective titer.

At day 56 (i.e. 28 days after the second injection) protective titerswere achieved in 0/3 of Group A, 2/3 of Group B and 9/9 of Group Crecipients of ALVAC-RG (vCP65) vaccine and persisted in all 10 HDCrecipients.

At day 56 the geometric mean titers were 0.05, 0.47, 4.4 and 11.5 IU/mlin groups A, B, C and HDC respectively.

At day 180, the rabies antibody titers had substantially decreased inall subjects but remained above the minimum protective titer of 0.5IU/ml in 5/10 HCD recipients and in 5/9 ALVAC-RG (vCP65) recipients; thegeometric mean titers were 0.51 and 0.45 IU/ml in groups HCD and C,respectively.

Antibodies to the Canarypox Virus (Table 17).

The pre-immune titers observed varied widely with titers varying from0.22 to 1.23 O.D. units despite the absence of any previous contact withcanary birds in those subjects with the highest titers. When defined asa greater than two-fold increase between preimmunization and post secondinjection titers, a seroconversion was obtained in 1/3 subjects in groupB and in 9/9 subjects in group C whereas no subject seroconverted ingroups A or HDC.

Booster Injection.

The vaccine was similarly well tolerated six months later, at the timeof the booster injection: fever was noted in 2/9 HDC booster recipientsand in 1/10 ALVAC-RG (vCP65) booster recipients. Local reactions werepresent in 5/9 recipients of HDC booster and in 6/10 recipients of theALVAC-RG (vCP65) booster.

Observations.

FIGS. 22A-22D shows graphs of rabies neutralizing antibody titers (RapidFluorescent Focus Inhibition Test or RFFIT, IU/ml): Booster effect ofHDC and vCP65 (10⁵.5 TCID₅₀) in volunteers previously immunized witheither the same or the alternate vaccine. Vaccines were given at days 0,28 and 180. Antibody titers were measured at days 0, 7, 28, 35, 56, 173,and 187 and 208.

As shown in FIGS. 22A-22D, the booster dose given resulted in a furtherincrease in rabies antibody titers in every subject whatever theimmunization scheme. However, the ALVAC-RG (vCP65) booster globallyelicited lower immune responses than the HDC booster and the ALVAC-RG(vCP65), ALVAC-RG (vCP65) - ALVAC-RG (vCP65) group had significantlylower titers than the three other groups. Similarly, the ALVAC-RG(vCP65) booster injection resulted in an increase in canarypox antibodytiters in 3/5 subjects who had previously received the HDC vaccine andin all five subjects previously immunized with ALVAC-RG (vCP65).

In general, none of the local side effects from administration of vCP65was indicative of a local replication of the virus. In particular,lesions of the skin such as those observed after injection of vaccinewere absent. In spite of the apparent absence of replication of thevirus, the injection resulted in the volunteers generating significantamounts of antibodies to both the canarypox vector and to the expressedrabies glycoprotein.

Rabies neutralizing antibodies were assayed with the Rapid FluorescentFocus Inhibition Test (RFFIT) which is known to correlate well with thesero neutralization test in mice. Of 9 recipients of 10⁵.5 TCID₅₀, fivehad low level responses after the first dose. Protective titers ofrabies antibodies were obtained after the second injection in allrecipients of the highest dose tested and even in 2 of the 3 recipientsof the medium dose. In this study, both vaccines were givensubcutaneously as usually recommended for live vaccines, but not for theinactivated HDC vaccine. This route of injection was selected as it bestallowed a careful examination of the injection site, but this couldexplain the late appearance of antibodies in HDC recipients: indeed,none of the HDC recipients had an antibody increase at day 7, whereas,in most studies where HDC vaccine is give intramuscularly a significantproportion of subjects do (Klietmann et al., Int'l Green Cross--Geneva,1981; Kuwert et al., Int'l Green Cross--Geneva, 1981). However, thisinvention is not necessarily limited to the subcutaneous route ofadministration.

The GMT (geometric mean titers) of rabies neutralizing antibodies waslower with the investigational vaccine than with the HDC controlvaccine, but still well above the minimum titer required for protection.The clear dose effect response obtained with the three dosages used inthis study suggest that a higher dosage might induce a strongerresponse. Certainly from this disclosure the skilled artisan can selectan appropriate dosage for a given patient.

The ability to boost the antibody response is another important resultof this Example; indeed, an increase in rabies antibody titers wasobtained in every subject after the 6 month dose whatever theimmunization scheme, showing that preexisting immunity elicited byeither the canarypox vector or the rabies glycoprotein had no blockingeffect on the booster with the recombinant vaccine candidate or theconventional HDC rabies vaccine. This contrasts findings of others withvaccinia recombinants in humans that immune response may be blocked bypre-existing immunity (Cooney et al., Lancet 1991, 337:567-72; Etingeret al., Vaccine 9:470-72, 1991).

Thus, this Example clearly demonstrates that a non-replicating poxviruscan serve as an immunizing vector in animals or humans, with all of theadvantages that replicating agents confer on the immune response, butwithout the safety problem created by a fully permissive virus.

                  TABLE 15                                                        ______________________________________                                        Reactions in the 5 days following vaccination                                          vCP65 dosage                                                                  (TCID50)         HDC                                                          10.sup.3.5                                                                           10.sup.4.5                                                                             10.sup.5.5                                                                             control                                     Injection  lst   2nd    1st 2nd  lst 2nd  lst 2nd                             ______________________________________                                        No. vaccinees                                                                            3     3      3   3    9   9    10  10                              temp >37.7° C.                                                                    0     0      0   0    0   1    1   0                               soreness   0     0      1   1    6   8    8   6                               redness    0     0      0   0    0   4    5   4                               induration 0     0      0   0    0   4    5   4                               ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        Rabies neutralizing antibodies (REFIT; IU/ml) Individual                      titers and geometric mean titers GMT)                                                    Days                                                               No.   TCID50/dose                                                                              0       7     28    35    56                                 ______________________________________                                         1    10.sup.3.5 <0.1    <0.1  <0.1  <0.1  0.2                                 3    10.sup.3.5 <0.1    <0.1  <0.1  <0.1  <0.1                                4    10.sup.3.5 <0.1    <0.1  <0.1  <0.1  <0.1                                     G.M.T.     <0.1    <0.1  <0.1  <0.1  <0.1                                6    10.sup.4.5 <0.1    <0.1  <0.1  <0.1  <0.1                                7    10.sup.4.5 <0.1    <0.1  <0.1  2.4   1.9                                10    10.sup.4.5 <0.1    <0.1  <0.1  1.6   1.1                                      G.M.T.     <0.1    <0.1  0.1   0.58  0.47                               11    10.sup.5.5 <0.1    <0.1  1.0   3.2   4.3                                13    10.sup.5.5 <0.1    <0.1  0.3   6.0   8.8                                14    10.sup.5.5 <0.1    <0.1  0.2   2.1   9.4                                17    10.sup.5.5 <0.1    <0.1  <0.1  1.2   2.5                                18    10.sup.5.5 <0.1    <0.1  0.7   8.3   12.5                               20    10.sup.5.5 <0.1    <0.1  <0.1  0.3   3.7                                21    10.sup.5.5 <0.1    <0.1  0.2   2.6   3.9                                23    10.sup.55  <0.1    <0.1  <0.1  1.7   4.2                                25    10.sup.55  <0.1    <0.1  <0.1  0.6   0.9                                      G.M.T.     <0.1    <0.1  0.16  1.9   4.4*                                2    HDC        <0.1    <0.1  0.8   7.1   7.2                                 5    HDC        <0.1    <0.1  9.9   12.8  18.7                                8    HDC        <0.1    <0.1  12.7  21.1  16.5                                9    HDC        <0.1    <0.1  6.0   9.9   14.3                               12    HDC        <0.1    <0.1  5.0   9.2   25.3                               15    HDC        <0.1    <0.1  2.2   5.2   8.6                                16    HDC        <0.1    <0.1  2.7   7.7   20.7                               19    HDC        <0.1    <0.1  2.6   9.9   9.1                                22    HDC        <0.1    <0.1  1.4   8.6   6.6                                24    HDC        <0.1    <0.1  0.8   5.8   4.7                                      G.M.T.     <0.1    <0.1  2.96  9.0   11.5*                              ______________________________________                                         *p = 0.007 student t test                                                

                  TABLE 17                                                        ______________________________________                                        Canarypox antibodies: ELISA Geometric Mean Titer*                             vCP65 dosage                                                                             Days                                                               TCID50/dose                                                                              0        7      28     35   56                                     ______________________________________                                        10.sup.3.5 0.69     ND     0.76   ND   0.68                                   10.sup.4.5 0.49     0.45   0.56   0.63 0.87                                   10.sup.5.5 0.38     0.38   0.77   1.42 1.63                                   HDC control                                                                              0.45     0.39   0.40   0.35 0.39                                   ______________________________________                                         *optical density at 1/25 dilution                                        

Example 23 Comparison of the LD₅₀ of ALVAC and NYVAC with VariousVaccinia Virus Strains

Mice.

Male outbred Swiss Webster mice were purchased from Taconic Farms(Germantown, N.Y.) and maintained on mouse chow and water ad libitumuntil use at 3 weeks of age ("normal" mice). Newborn outbred SwissWebster mice were of both sexes and were obtained following timedpregnancies performed by Taconic Farms. All newborn mice used weredelivered within a two day period.

Viruses.

ALVAC was derived by plaque purification of a canarypox virus populationand was prepared in primary chick embryo fibroblast cells (CEF).Following purification by centrifugation over sucrose density gradients,ALVAC was enumerated for plaque forming units in CEF cells. The WR(L)variant of vaccinia virus was derived by selection of large plaquephenotypes of WR (Panicali et al., 1981). The Wyeth New York State Boardof Health vaccine strain of vaccinia virus was obtained fromPharmaceuticals Calf Lymph Type vaccine Dryvax, control number 302001B.Copenhagen strain vaccinia virus VC-2 was obtained from InstitutMerieux, France. Vaccinia virus strain NYVAC was derived from CopenhagenVC-2. All vaccinia virus strains except the Wyeth strain were cultivatedin Vero African green monkey kidney cells, purified by sucrose gradientdensity centrifugation and enumerated for plaque forming units on Verocells. The Wyeth strain was grown in CEF cells and enumerated in CEFcells.

Inoculations.

Groups of 10 normal mice were inoculated intracranially (ic) with 0.05ml of one of several dilutions of virus prepared by 10-fold seriallydiluting the stock preparations in sterile phosphate-buffered saline. Insome instances, undiluted stock virus preparation was used forinoculation.

Groups of 10 newborn mice, 1 to 2 days old, were inoculated ic similarlyto the normal mice except that an injection volume of 0.03 ml was used.

All mice were observed daily for mortality for a period of 14 days(newborn mice) or 21 days (normal mice) after inoculation. Mice founddead the morning following inoculation were excluded due to potentialdeath by trauma.

The lethal dose required to produce mortality for 50% of theexperimental population (LD₅₀) was determined by the proportional methodof Reed and Muench.

Comparison of the LD₅₀ of ALVAC and NYVAC with Various Vaccinia VirusStrains for Normal, Young Outbred Mice by the ic Route.

In young, normal mice, the virulence of NYVAC and ALVAC were severalorders of magnitude lower than the other vaccinia virus strains tested(Table 18). NYVAC and ALVAC were found to be over 3,000 times lessvirulent in normal mice than the Wyeth strain; over 12,500 times lessvirulent than the parental VC-2 strain; and over 63,000,000 times lessvirulent than the WR(L) variant. These results would suggest that NYVACis highly attenuated compared to other vaccinia strains, and that ALVACis generally nonvirulent for young mice when administeredintracranially, although both may cause mortality in mice at extremelyhigh doses (3.85×10⁸ PFUs, ALVAC and 3×10⁸ PFUS, NYVAC) by anundetermined mechanism by this route of inoculation.

Comparison of the LD₅₀ of ALVAC and NYVAC with Various Vaccinia VirusStrains for Newborn Outbred Mice by the ic Route.

The relative virulence of 5 poxvirus strains for normal, newborn micewas tested by titration in an intracranial (ic) challenge model system(Table 19). With mortality as the endpoint, LD₅₀ values indicated thatALVAC is over 100,000 times less virulent than the Wyeth vaccine strainof vaccinia virus; over 200,000 times less virulent than the CopenhagenVC-2 strain of vaccinia virus; and over 25,000,000 times less virulentthan the WR-L variant of vaccinia virus. Nonetheless, at the highestdose tested, 6.3×10⁷ PFUs, 100% mortality resulted. Mortality rates of33.3% were observed at 6.3×10⁶ PFUs. The cause of death, while notactually determined, was not likely of toxicological or traumatic naturesince the mean survival time (MST) of mice of the highest dosage group(approximately 6.3 LD₅₀) was 6.7±1.5 days. When compared to WR(L) at achallenge dose of 5 LD₅₀, wherein MST is 4.8±0.6 days, the MST of ALVACchallenged mice was significantly longer (P=0.001).

Relative to NYVAC, Wyeth was found to be over 15,000 times morevirulent; VC-2, greater than 35,000 times more virulent; and WR(L), over3,000,000 times more virulent. Similar to ALVAC, the two highest dosesof NYVAC, 6×10⁸ and 6×10⁷ PFUs, caused 100% mortality. However, the MSTof mice challenged with the highest dose, corresponding to 380 LD₅₀, wasonly 2 days (9 deaths on day 2 and 1 on day 4). In contrast, all micechallenged with the highest dose of WR-L, equivalent to 500 LD₅₀,survived to day 4.

                  TABLE 18                                                        ______________________________________                                        Calculated 50% Lethal Dose for mice by various                                vaccinia virus strains and for canarypox virus                                (ALVAC) by the ic route.                                                      POXVIRUS      CALCULATED                                                      STRAIN        LD.sub.50 (PFUs)                                                ______________________________________                                        WR(L)         2.5                                                             VC-2          1.26 × 10.sup.4                                           WYETH         5.00 × 10.sup.4                                           NYVAC         1.58 × 10.sup.8                                           ALVAC         1.58 × 10.sup.8                                           ______________________________________                                    

                  TABLE 19                                                        ______________________________________                                        Calculated 50% Lethal Dose for newborn mice by                                various vaccinia virus strains and for canarypox                              virus (ALVAC) by the ic route.                                                POXVIRUS      CALCULATED                                                      STRAIN        LD.sub.50 (PFUs)                                                ______________________________________                                        WR(L)         0.4                                                             VC-2          0.1                                                             WYETH         1.6                                                             NYVAC         1.58 × 10.sup.6                                           ALVAC         1.00 × 10.sup.7                                           ______________________________________                                    

Example 24 Evaluation of NYVAC (vP866) and NYVAC-RG (vP879)

Immunoprecipitations.

Preformed monolayers of avian or non-avian cells were inoculated with 10pfu per cell of parental NYVAC (vP866) or NYVAC-RG (vP879) virus. Theinoculation was performed in EMEM free of methionine and supplementedwith 2% dialyzed fetal bovine serum. After a one hour incubation, theinoculum was removed and the medium replaced with EMEM (methionine free)containing 20 μCi/ml of ³⁵ S-methionine. After an overnight incubationof approximately 16 hours, cells were lysed by the addition of Buffer A(1% Nonidet P-40, 10 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 0.01% sodiumazide, 500 units per ml of aprotinin, and 0.02% phenyl methyl sulfonylfluoride). Immunoprecipitation was performed using a rabies glycoproteinspecific monoclonal antibody designated 24-3F10 supplied by Dr. C.Trimarchi, Griffith Laboratories, New York State Department of Health,Albany, N.Y., and a rat anti-mouse conjugate obtained from BoehringerMannheim Corporation (Cat. #605-500). Protein A Sepharose CL-48 obtainedfrom Pharmacia LKB Biotechnology Inc., Piscataway, N.J., was used as asupport matrix. Immunoprecipitates were fractionated on 10%polyacrylamide gels according to the method of Dreyfuss et. al. (1984).Gels were fixed, treated for fluorography with 1M Na-salicylate for onehour, and exposed to Kodak XAR-2 film to visualize theimmunoprecipitated protein species.

Sources of Animals.

New Zealand White rabbits were obtained from Hare-Marland (Hewitt,N.J.). Three week old male Swiss Webster outbred mice, timed pregnantfemale Swiss Webster outbred mice, and four week old Swiss Webster nude(nu⁺ nu⁺) mice were obtained from Taconic Farms, Inc. (Germantown,N.Y.). All animals were maintained according to NIH guidelines. Allanimal protocols were approved by the institutional IACUC. When deemednecessary, mice which were obviously terminally ill were euthanized.

Evaluation of Lesions in Rabbits.

Each of two rabbits was inoculated intradermally at multiple sites with0.1 ml of PBS containing 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ pfu of each testvirus or with PBS alone. The rabbits were observed daily from day 4until lesion resolution. Indurations and ulcerations were measured andrecorded.

Virus Recovery from Inoculation Sites.

A single rabbit was inoculated intradermally at multiple sites of 0/1 mlof PBS containing 10⁶, 10⁷, or 10⁸ pfu of each test virus or with PBSalone. After 11 days, the rabbit was euthanized and skin biopsyspecimens taken from each of the inoculation sites were asepticallyprepared by mechanical disruption and indirect sonication for virusrecovery. Infectious virus was assayed by plaque titration on CEFmonolayers.

Virulence in Mice.

Groups of ten mice, or five in the nude mice experiment, were inoculatedip with one of several dilutions of virus in 0.5 ml of sterile PBS.Reference is also made to Example 23.

Cyclophosphamide (CY) Treatment.

Mice were injected by the ip route with 4 mg (0.02 ml) of CY (SIGMA) onday -2, followed by virus injection on day 0. On the following days postinfection, mice were injected ip with CY: 4 mg on day 1; 2 mg on days 4,7 and 11; 3 mg on days 14, 18, 21, 25 and 28. Immunosuppression wasindirectly monitored by enumerating white blood cells with a CoulterCounter on day 11. The average white blood cell count was 13,500 cellsper μl for untreated mice (n=4) and 4,220 cells per μl for CY-treatedcontrol mice (n=5).

Calculation of LD₅₀.

The lethal dose required to produce 50% mortality (LD₅₀) was determinedby the proportional method of Reed and Muench (Reed and Muench 1938).

Potency Testing of NYVAC-RG in Mice.

Four to six week old mice were inoculated in the footpad with 50 to 100μl of a range of dilutions (2.0-8.0 log₁₀ tissue culture infective dose50% (TCID₅₀)) of either VV-RG (Kieny et al., 1984), ALVAC-RG (Taylor etal., 1991b), or the NYVAC-RG. Each group consisted of eight mice. At 14days post-vaccination, the mice were challenged by intracranialinoculation with 15 LD₅₀ of the rabies virus CVS strain (0.03 ml). Onday 28, surviving mice were counted and protective does 50% (PD₅₀)calculated.

Derivation of NYVAC (vP866).

The NYVAC strain of vaccinia virus was generated from VC-2, a plaquecloned isolate of the COPENHAGEN vaccine strain. To generate NYVAC fromVC-2, eighteen vaccinia ORFs, including a number of viral functionsassociated with virulence, were precisely deleted in a series ofsequential manipulations as described earlier in this disclosure. Thesedeletions were constructed in a manner designed to prevent theappearance of novel unwanted open reading frames. FIG. 19 schematicallydepicts the ORFs deleted to generate NYVAC. At the top of FIG. 19 isdepicted the HindIII restriction map of the vaccinia virus genome (VC-2plaque isolate, COPENHAGEN strain). Expanded are the six regions of VC-2that were sequentially deleted in the generation of NYVAC. The deletionswere described earlier in this disclosure (Examples 13 through 18).Below such deletion locus is listed the ORFs which were deleted fromthat locus, along with the functions or homologies and molecular weightof their gene products.

Replication Studies of NYVAC and ALVAC on Human Tissue Cell Lines.

In order to determine the level of replication of NYVAC strain ofvaccinia virus (vP866) in cells of human origin, six cell lines wereinoculated at an input multiplicity of 0.1 pfu per cell under liquidculture and incubated for 72 hours. The COPENHAGEN parental clone (VC-2)was inoculated in parallel. Primary chick embryo fibroblast (CEF) cells(obtained from 10-11 day old embryonated eggs of SPF origin, Spafas,Inc., Storrs, Conn.) were included to represent a permissive cellsubstrate for all viruses. Cultures were analyzed on the basis of twocriteria: the occurrence of productive viral replication and expressionof an extrinsic antigen.

The replication potential of NYVAC in a number of human derived cellsare shown in Table 20. Both VC-2 and NYVAC are capable of productivereplication in CEF cells, although NYVAC with slightly reduced yields.VC-2 is also capable of productive replication in the six human derivedcell lines tested with comparable yields except in the EBV transformedlymphoblastoid cell line JT-1 (human lymphoblastoid cell linetransformed with Epstein-Barr virus, see Rickinson et al., 1984). Incontract, NYVAC is highly attenuated in its ability to productivelyreplicate in any of the human derived cell lines tested. Small increasesof infectious virus above residual virus levels were obtained fromNYVAC-infected MRC-5 (ATCC #CCL171, human embryonic lung origin),DETROIT 532 (ATCC #CCL54, human foreskin, Downs Syndrome), HEL 299 (ATCC#CCL137, human embryonic lung cells) and HNK (human neonatal kidneycells, Whittiker Bioproducts, Inc. Walkersville, Md., Cat #70-151)cells. Replication on these cell lines was significantly reduced whencompared to virus yields obtained from NYVAC-infected CEF cells or withparental VC-2 (Table 20). It should be noted that the yields at 24 hoursin CEF cells for both NYVAC and VC-2 is equivalent to the 72-hour yield.Allowing the human cell line cultures to incubate an additional 48 hours(another two viral growth cycles) may, therefore, have amplified therelative virus yield obtained.

Consistent with the low levels of virus yields obtained in thehuman-derived cell lines, MRC-5 and DETROIT 532, detectable but reducedlevels of NYVAC-specific DNA accumulation were noted. The level of DNAaccumulation in the MRC-5 and DETROIT 532 NYVAC-infected cell linesrelative to that observed in NYVAC-infected CEF cells paralleled therelative virus yields. NYVAC-specific viral DNA accumulation was notobserved in any of the other human-derived cells.

An equivalent experiment was also performed using the avipox virus,ALVAC. The results of virus replication are also shown in Table 20. Noprogeny virus was detectable in any of the human cell lines consistentwith the host range restriction of canarypox virus to avian species.Also consistent with a lack of productive replication of ALVAC in thesehuman-derived cells is the observation that no ALVAC-specific DNAaccumulation was detectable in any of the human-derived cell lines.

Expression of Rabies Glycoprotein by NYVAC-RG (vP879) in Human Cells.

In order to determine whether efficient expression of a foreign genecould be obtained in the absence of significant levels of productiveviral replication, the same cell lines were inoculated with the NYVACrecombinant expressing the rabies virus glycoprotein (vP879, Example 19)in the presence of ³⁵ S-methionine. Immunoprecipitation of the rabiesglycoprotein was performed from the radiolabelled culture lysate using amonoclonal antibody specific for the rabies glycoprotein.Immunoprecipitation of a 67 kDa protein was detected consistent with afully glycosylated form of the rabies glycoprotein. No serologicallycrossreactive product was detected in uninfected or parental NYVACinfected cell lysates. Equivalent results were obtained with all otherhuman cells analyzed.

Inoculations on the Rabbit Skin.

The induction and nature of skin lesions on rabbits followingintradermal (id) inoculations has been previously used as a measure ofpathogenicity of vaccinia virus strains (Buller et al., 1988; Child etal., 1990; Fenner, 1958, Flexner et al., 1987; Ghendon and Chernos1964). Therefore, the nature of lesions associated with id inoculationswith the vaccinia strains WR (ATCC #VR119 plaque purified on CV-1 cells,ATCC #CCL70, and a plaque isolate designated L variant, ATCC #VR2035selected, as described in Panicali et al., 1981)), WYETH (ATCC #VR325marketed as DRYVAC by Wyeth Laboratories, Marietta, Pa.), COPENHAGEN(VC-2), and NYVAC was evaluated by inoculation of two rabbits (A069 andA128). The two rabbits displayed different overall sensitivities to theviruses, with rabbit A128 displaying less severe reactions than rabbitA069. In rabbit A128, lesions were relatively small and resolved by 27days post-inoculation. On rabbit A069, lesions were intense, especiallyfor the WR inoculation sites, and resolved only after 49 days. Intensityof the lesions was also dependent on the location of the inoculationsites relative to the lymph drainage network. In particular, all siteslocated above the backspine displayed more intense lesions and requiredlonger times to resolve the lesions located on the flanks. All lesionswere measured daily from day 4 to the disappearance of the last lesion,and the means of maximum lesion size and days to resolution werecalculated (Table 21). No local reactions were observed from sitesinjected with the control PBS. Ulcerative lesions were observed at sitesinjected with WR, VC-2 and WYETH vaccinia virus strains. Significantly,no induration or ulcerative lesions were observed at sites ofinoculation with NYVAC.

Persistence of Infectious Virus at the Site of Inoculation.

To assess the relative persistence of these viruses at the site ofinoculation, a rabbit was inoculated intradermally at multiple siteswith 0.1 ml PBS containing 10⁶, 10⁷ or 10⁸ pfu of VC-2, WR, WYETH orNYVAC. For each virus, the 10⁷ pfu dose was located above the backspine,flanked by the 10⁶ and 10⁸ doses. Sites of inoculation were observeddaily for 11 days. WR elicited the most intense response, followed byVC-2 and WYETH (Table 22). Ulceration was first observed at day 9 for WRand WYETH and day 10 for VC-2. Sites inoculated with NYVAC or controlPBS displayed no induration or ulceration. At day 11 after inoculation,skin samples from the sites of inoculation were excised, mechanicallydisrupted, and virus was titrated on CEF cells. The results are shown inTable 22. In no case was more virus recovered at this timepoint than wasadministered. Recovery of vaccinia strain, WR, was approximately 10⁶ pfuof virus at each site irrespective of amount of virus administered.Recovery of vaccinia strains WYETH and VC-2 was 10³ to 10⁴ pfuregardless of amount administered. No infectious virus was recoveredfrom sites inoculated with NYVAC.

Inoculation of Genetically or Chemically Immune Deficient Mice.

Intraperitoneal inoculation of high doses of NYVAC (5×10⁸ pfu) or ALVAC(10⁹ pfu) into nude mice caused no deaths, no lesions, and no apparentdisease through the 100 day observation period. In contrast, miceinoculated with WR (10³ to 10⁴ pfu), WYETH (5×10⁷ or 5×10⁸ pfu) or VC-2(10⁴ to 10⁹ pfu) displayed disseminated lesions typical of poxvirusesfirst on the toes, then on the tail, followed by severe orchitis in someanimals. In mice infected with WR or WYETH, the appearance ofdisseminated lesions generally led to eventual death, whereas most miceinfected with VC-2 eventually recovered. Calculated LD₅₀ values aregiven in Table 23.

In particular, mice inoculated with VC-2 began to display lesions ontheir toes (red papules) and 1 to 2 days later on the tail. Theselesions occurred between 11 and 13 days post-inoculation (pi) in micegiven the highest doses (10⁹, 10⁸, 10⁷ and 10⁶ pfu), on day 16 pi inmice given 10⁵ pfu and on day 21 pi in mice given 10⁴ pfu. No lesionswere observed in mice inoculated with 10³ and 10² pfu during the 100 dayobservation period. Orchitis was noticed on day 23 pi in mice given 10⁹and 10⁸ pfu, and approximately 7 days later in the other groups (10⁷ to10⁴ pfu). Orchitis was especially intense in the 10⁹ and 10⁸ pfu groupsand, although receding, was observed until the end of the 100 dayobservation period. Some pox-like lesions were noticed on the skin of afew mice, occurring around 30-35 days pi. Most pox lesions healednormally between 60-90 days pi. Only one mouse died in the groupinoculated with 10⁹ pfu (Day 34 pi) and one mouse died in the groupinoculated with 10⁸ pfu (Day 94 pi). No other deaths were observed inthe VC-2 inoculated mice.

Mice inoculated with 10⁴ pfu of the WR strain of vaccinia started todisplay pox lesions on Day 17 pi. These lesions appeared identical tothe lesions displayed by the VC-2 injected mice (swollen toes, tail).Mice inoculated with 10³ pfu of the WR strain did not develop lesionsuntil 34 days pi. Orchitis was noticed only in the mice inoculated withthe highest dose of WR (10⁴ pfu). During the latter stages of theobservation period, lesions appeared around the mouth and the micestopped eating. All mice inoculated with 10⁴ pfu of WR died or wereeuthanized when deemed necessary between 21 days and 31 days pi. Fourout of the 5 mice injected with 10³ pfu of WR died or were euthanizedwhen deemed necessary between 35 days and 57 days pi. No deaths wereobserved in mice inoculated with lower doses of WR (1 to 100 pfu).

Mice inoculated with the WYETH strain of vaccinia virus at higher doses5×10⁷ and 5×10⁸ pfu) showed lesions on toes and tails, developedorchitis, and died. Mice injected with 5×10⁶ pfu or less of WYETH showedno signs of disease or lesions.

As shown in Table 23, CY-treated mice provided a more sensitive modelfor assaying poxvirus virulence than did nude mice. LD₅₀ values for theWR, WYETH, and VC-2 vaccinia virus strains were significantly lower inthis model system than in the nude mouse model. Additionally, lesionsdeveloped in mice injected with WYETH, WR and VC-2 vaccinia viruses, asnoted below, with higher doses of each virus resulting in more rapidformation of lesions. As was seen with nude mice, CY-treated miceinjected with NYVAC or ALVAC did not develop lesions. However, unlikenude mice, some deaths were observed in CY-treated mice challenged withNYVAC or ALVAC, regardless of the dose. These random incidences aresuspect as to the cause of death.

Mice injected with all doses of WYETH (9.5×10⁴ to 9.5×10⁸ pfu) displayedpox lesions on their tail and/or on their toes between 7 and 15 days pi.In addition, the tails and toes were swollen. Evolution of lesions onthe tail was typical of pox lesions with formation of a papule,ulceration and finally formation of a scab. Mice inoculated with alldoses of VC-2 (1.65×10⁵ to 1.65×10⁹) also developed pox lesions on theirtails and/or their toes analogous to those of WYETH injected mice. Theselesions were observed between 7-12 days post inoculation. No lesionswere observed on mice injected with lower doses of WR virus, althoughdeaths occurred in these groups.

Potency Testing of NYVAC-RG.

In order to determine that attenuation of the COPENHAGEN strain ofvaccinia virus had been effected without significantly altering theability of the resulting NYVAC strain to be a useful vector, comparativepotency tests were performed. In order to monitor the immunogenicpotential of the vector during the sequential genetic manipulationsperformed to attenuate the virus, a rabiesvirus glycoprotein was used asa reporter extrinsic antigen. The protective efficacy of the vectorsexpressing the rabies glycoprotein gene was evaluated in the standardNIH mouse potency test for rabies (Seligmann, 1973). Table 24demonstrates that the PD₅₀ values obtained with the highly attenuatedNYVAC vector are identical to those obtained using a COPENHAGEN-basedrecombinant containing the rabies glycoprotein gene in the tk locus(Kieny et al., 1984) and similar to PD₅₀ values obtained with ALVAC-RG,a canarypox based vector restricted to replication to avian species.

Observations.

NYVAC, deleted of known virulence genes and having restricted in vitrogrowth characteristics, was analyzed in animal model systems to assessits attenuation characteristics. These studies were performed incomparison with the neurovirulent vaccinia virus laboratory strain, WR,two vaccinia virus vaccine strains, WYETH (New York City Board ofHealth) and COPENHAGEN (VC-2), as well as with a canarypox virus strain,ALVAC (See also Example 23). Together, these viruses provided a spectrumof relative pathogenic potentials in the mouse challenge model and therabbit skin model, with WR being the most virulent strain, WYETH andCOPENHAGEN (VC-2) providing previously utilized attenuated vaccinestrains with documented characteristics, and ALVAC providing an exampleof a poxvirus whose replication is restricted to avian species. Resultsfrom these in vivo analyses clearly demonstrate the highly attenuatedproperties of NYVAC relative to the vaccinia virus strains, WR, WYETHand COPENHAGEN (VC-2) (Tables 18-24). Significantly, the LD₅₀ values forNYVAC were comparable to those observed with the avian host restrictedavipoxvirus, ALVAC. Deaths due to NYVAC, as well as ALVAC, were observedonly when extremely high doses of virus were administered via theintracranial route (Example 23, Tables 18, 19, 23). It has not yet beenestablished whether these deaths were due to nonspecific consequences ofinoculation of a high protein mass. Results from analyses inimmunocompromised mouse models (nude and CY-treated) also demonstratethe relatively high attenuation characteristics of NYVAC, as compared toWR, WYETH and COPENHAGEN strains (Tables 21 and 22). Significantly, noevidence of disseminated vaccinia infection or vaccinial disease wasobserved in NYVAC-inoculated animals or ALVAC-inoculated animals overthe observation period. The deletion of multiple virulence-associatedgenes in NYVAC shows a synergistic effect with respect to pathogenicity.Another measure of the innocuity of NYVAC was provided by theintradermal administration on rabbit skin (Tables 21 and 22).Considering the results with ALVAC, a virus unable to replicate innonavian species, the ability to replicate at the site of inoculation isnot the sole correlate with reactivity, since intradermal inoculation ofALVAC caused areas of induration in a dose dependent manner. Therefore,it is likely that factors other than the replicative capacity of thevirus contribute to the formation of the lesions. Deletion of genes inNYVAC prevents lesion occurrence.

Together, the results in this Example and in foregoing Examples,including Example 23, demonstrate the highly attenuated nature of NYVACrelative to WR, and the previously utilized vaccinia virus vaccinestrains, WYETH and COPENHAGEN. In fact, the pathogenic profile of NYVAC,in the animal model systems tested, was similar to that of ALVAC, apoxvirus known to productively replicate only in avian species. Theapparently restricted capacity of NYVAC to productively replicate oncells derived from humans (Table 20) and other species, including themouse, swine, dog and horse, provides a considerable barrier that limitsor prevents potential transmission to unvaccinated contacts or to thegeneral environment in addition to providing a vector with reducedprobability of dissemination within the vaccinated individual.

Significantly, NYVAC-based vaccine candidates have been shown to beefficacious. NYVAC recombinants expressing foreign gene products from anumber of pathogens have elicited immunological responses towards theforeign gene products in several animal species, including primates. Inparticular, a NYVAC-based recombinant expressing the rabies glycoproteinwas able to protect mice against a lethal rabies challenge. The potencyof the NYVAC-based rabies glycoprotein recombinant was comparable to thePD₅₀ value for a COPENHAGEN-based recombinant containing the rabiesglycoprotein in the tk locus (Table 24). NYVAC-based recombinants havealso been shown to elicit measles virus neutralizing antibodies inrabbits and protection against pseudorabies virus and Japaneseencephalitis virus challenge in swine. The highly attenuated NYVACstrain confers safety advantages with human and veterinary applications(Tartaglia et al., 1990). Furthermore, the use of NYVAC as a generallaboratory expression vector system may greatly reduce the biologicalhazards associated with using vaccinia virus.

By the following criteria, the results of this Example and the Examplesherein, including Example 23, show NYVAC to be highly attenuated: a) nodetectable induration or ulceration at site of inoculation (rabbitskin); b) rapid clearance of infectious virus from intradermal site ofinoculation (rabbit skin); c) absence of testicular inflammation (nudemice); d) greatly reduced virulence (intracranial challenge, boththree-week old and newborn mice); e) greatly reduced pathogenicity andfailure to disseminate in immunodeficient subjects (nude andcyclophosphamide treated mice); and f) dramatically reduced ability toreplicate on a variety of human tissue culture cells. Yet, in spite ofbeing highly attenuated, NYVAC, as a vector, retains the ability toinduce strong immune responses to extrinsic antigens.

                  TABLE 20                                                        ______________________________________                                        Replication of COPENHAGEN (VC-2), NYVAC and ALVAC                             in avian or human derived cell lines                                                 Hours                                                                         post-  Yield.sup.a        %                                            Cells    infection                                                                              VC-2     NYVAC  ALVAC  Yield                                ______________________________________                                        CEF       0        3.8.sup.b                                                                             3.7    4.5                                                  24       8.3      7.8    6.6                                                  48       8.6      7.9    7.7                                                  72       8.3      7.7    7.5    25                                             72A.sup.c                                                                             <1.4     1.8    3.1                                         MRC-5     0       3.8      3.8    4.7                                                  72       7.2      4.6    3.8    0.25                                           72A     2.2      2.2    3.7                                         WISH*     0       3.4      3.4    4.3                                                  72       7.6      2.2    3.1    0.000                                                                         4                                              72A      --.sup.d                                                                              1.9    2.9                                         DETROIT   0       3.8      3.7    4.4                                                  72       7.2      5.4    3.4    1.6                                            72A     1.7      1.7    2.9                                         HEL       0       3.8      3.5    4.3                                                  72       7.5      4.6    3.3    0.125                                          72A     2.5      2.1    3.6                                         JT-1      0       3.1      3.1    4.1                                                  72       6.5      3.1    4.2    0.039                                          72A     2.4      2.1    4.4                                         HNK       0       3.8      3.7    4.7                                                  72       7.6      4.5    3.6    0.079                                          72A     3.1      2.7    3.7                                         ______________________________________                                         .sup.a Yield of NYVAC at 72 hours postinfection expressed as a percentage     of yield of VAC2 after 72 hours on the same cell line.                        .sup.b Titer expressed as LOG.sub.50  pfu per ml.                             .sup.c Sample was incubated in the presence of 40 μg/ml of cytosine        arabinoside.                                                                  .sup.d Not determined.                                                        *ATCC #CCL25 Human amnionic cells.                                       

                  TABLE 21                                                        ______________________________________                                        Induration and ulceration at the site of                                      intradermal inoculation of the rabbit skin                                    VIRUS          INDURATION   ULCERATION                                        STRAIN   DOSE.sup.a                                                                              Size.sup.b                                                                            Days.sup.c                                                                           Size Days                                   ______________________________________                                        WR       10.sup.4  386     30      88  30                                              10.sup.5  622     35     149  32                                              10.sup.6  1057    34     271  34                                              10.sup.7  877     35     204  35                                              10.sup.8  581     25      88  26                                     WYETH    10.sup.4   32      5      --.sup.d                                                                          --                                              10.sup.5  116     15     --   --                                              10.sup.6  267     17      3   15                                              10.sup.7  202     17      3   24                                              10.sup.8  240     29      12  31                                     VC-2     10.sup.4   64      7     --   --                                              10.sup.5   86      8     --   --                                              10.sup.6  136     17     --   --                                              10.sup.7  167     21      6   10                                              10.sup.8  155     32      6    8                                     NYVAC    10.sup.4  --      --     --   --                                              10.sup.5  --      --     --   --                                              10.sup.6  --      --     --   --                                              10.sup.7  --      --     --   --                                              10.sup.8  --      --     --   --                                     ______________________________________                                         .sup.a pfu of indicated vaccinia virus in 0.1 ml PBS inoculated               intradermally into one sight.                                                 .sup.b mean maximum size of lesions (mm.sup.2)                                .sup.c mean time after inoculation for complete healing of lesion.            .sup.d no lesions discernable.                                           

                  TABLE 22                                                        ______________________________________                                        Persistence of poxviruses at the site of                                      intradermal inoculation                                                                          Total Virus                                                Virus         Inoculum Dose                                                                            Recovered                                            ______________________________________                                        WR             8.0.sup.a 6.14                                                               7.0        6.26                                                               6.0        6.21                                                 WYETH         8.0        3.66                                                               7.0        4.10                                                               6.0        3.59                                                 VC-2          8.0        4.47                                                               7.0        4.74                                                               6.0        3.97                                                 NYVAC         8.0        0                                                                  7.0        0                                                                  6.0        0                                                    ______________________________________                                         .sup.a expressed as log.sub.10  pfu.                                     

                  TABLE 23                                                        ______________________________________                                        Virulence studies in immunocompromised mice                                             LD.sub.50 .sup.a                                                    Poxvirus              Cyclophosphamide                                        Strain      Nude mice treated mice                                            ______________________________________                                        WR          422       42                                                      VC-2        >10.sup.9 <1.65 × 10.sup.5                                  WYETW        1.58 × 10.sup.7                                                                   1.83 × 10.sup.6                                  NYVAC       >5.50 × 10.sup.8                                                                   7.23 × 10.sup.8                                  ALVAC       >10.sup.9  ≧5.00 × 10.sup.8b                         ______________________________________                                         .sup.a Calculated 50% lethal dose (pfu) for nude or cyclophosphamide          treated mice by the indicated vaccinia viruses and for ALVAC by               intraperitoneal route.                                                        .sup.b 5 out of 10 mice died at the highest dose of 5 × 10.sup.8        pfu.                                                                     

                  TABLE 24                                                        ______________________________________                                        Comparative efficacy of NYVAC-RG and ALVAC-RG in mice                                 Recombinant                                                                           PD.sub.50 .sup.a                                              ______________________________________                                                VV-RG   3.74                                                                  ALVAC-RG                                                                              3.86                                                                  NYVAC-RG                                                                              3.70                                                          ______________________________________                                         .sup.a Four to six week old mice were inoculated in the footpad with          50-100 μl of a range of dilutions (2.0-8.0 log.sub.10  tissue culture      infection dose 50% (TCID.sub.50) of either the VVRG (Kieny et al., 1984),     ALVACRG (vCP65) or NYVACRG (vP879). At day 14, mice of each group were        challenged by intracranial inoculation of 30 μl of a live CVS strain       rabies virus corresponding to 15 lethal dose 50% (LD.sub.50) per mouse. A     day 28, surviving mice were counted and a protective do  # se 50%             (PD.sub.50) was calculated.                                              

Example 25 Construction of Trovac Recombinants Expressing theHemagglutinin Glycoproteins of Avian Influenza Viruses

This Example describes the development of fowlpox virus recombinantsexpressing the hemagglutinin genes of three serotypes of avian influenzavirus.

Cells and Viruses.

Plasmids containing cDNA clones of the H4, H5 and H7 hemagglutinin geneswere obtained from Dr. Robert Webster, St. Jude Children's ResearchHospital, Memphis, Tenn. The strain of FPV designated FP-1 has beendescribed previously (Taylor et al., 1988a, b). It is a vaccine strainuseful in vaccination of day old chickens. The parental virus strainDuvette was obtained in France as a fowlpox scab from a chicken. Thevirus was attenuated by approximately 50 serial passages in chickenembryonated eggs followed by 25 passages on chick embryo fibroblast(CEF) cells. This virus was obtained in September 1980 by Rhone Merieux,Lyon, France, and a master viral seed established. The virus wasreceived by Virogenetics in September 1989, where it was subjected tofour successive plaque purifications. One plaque isolate was furtheramplified in primary CEF cells and a stock virus, designated as TROVAC,was established. The stock virus used in the in vitro recombination testto produce TROVAC-AIH5 (vFP89) and TROVAC-AIH4 (vFP92) had been furtheramplified though 8 passages in primary CEF cells. The stock virus usedto produce TROVAC-AIH7 (vFP100) had been further amplified through 12passages in primary CEF cells.

Construction of Fowlpox Insertion Plasmid at F8 Locus.

Plasmid pRW731.15 contains a 10 kbp PvuII-PvuII fragment cloned fromTROVAC genomic DNA. The nucleotide sequence was determined on bothstrands for a 3659 bp PvuII-EcoRV fragment. This sequence is shown inFIG. 20 (SEQ ID NO:72). The limits of an open reading frame designatedin this laboratory as F8 were determined within this sequence. The openreading frame is initiated at position 495 and terminates at position1887. A deletion was made from position 779 to position 1926, asdescribed below.

Plasmid pRW761 is a sub-clone of pRW731.15 containing a 2430 bpEcoRV-EcoRV fragment. Plasmid pRW761 was completely digested with XbaIand partially digested with SspI. A 3700 bp XbaI-SspI band was isolatedand ligated with the annealed double-stranded oligonucleotides JCA017(SEQ ID NO:60) and JCA018 (SEQ ID NO:61).

    JCA017                                                                            (SEQ ID NO:60):                                                                         5' CTAGACACTTTATGTTTTTTAATATCCGGTCTT                                          AAAAGCTTCCCGGGGATCCTTATACGGGGAATAAT 3'                          JCA018                                                                            (SEQ ID NO:61):                                                                         5' ATTATTCCCCGTATAAGGATCCCCCGGGAA                                             GCTTTTAAGACCGGATATTAAAAAACATAAAGTGT 3'                      

The plasmid resulting from this ligation was designated pJCA002. PlasmidpJCA004 contains a non-pertinent gene linked to the vaccinia virus H6promoter in plasmid pJCA002. The sequence of the vaccinia virus H6promoter has been previously described (Taylor et al., 1988a, b; Guo etal. 1989; Perkus et al., 1989). Plasmid pJCA004 was digested with EcoRVand BamHI which deletes the non-pertinent gene and a portion of the 3'end of the H6 promoter. Annealed oligonucleotides RW178 (SEQ ID NO:73)and RW179 (SEQ ID NO:74) were cut with EcoRV and BamHI and insertedbetween the EcoRV and BamHI sites of JCA004 to form pRW846.

    RW178                                                                              (SEQ ID NO:73):                                                                        5' TCATTATCGCGATATCCGTGTTAACTAGCTA                                            GCTAATTTTTATTTCCCGGGATCCTTATCA 3'                               RW179                                                                              (SEQ ID NO:74):                                                                        5' GTATAAGGATCCCGGGAATAAAAATTAGCT                                             AGCTAGTTAACACGGATATCGCGATAATGA 3'                           

Plasmid pRW846 therefore contains the H6 promoter 5' of EcoRV in thede-ORFed F8 locus. The HincII site 3' of the H6 promoter in pRW846 isfollowed by translation stop codons, a transcriptional stop sequencerecognized by vaccinia virus early promoters (Yuen et al., 1987) and aSmaI site.

Construction of Fowlpox Insertion Plasmid at F7 Locus.

The original F7 non-de-ORFed insertion plasmid, pRW731.13, contained a5.5 kb FP genomic PvuII fragment in the PvuII site of pUC9. Theinsertion site was a unique HincII site within these sequences. Thenucleotide sequence shown in FIG. 21 (SEQ ID NO:75) was determined for a2356 bp region encompassing the unique HincII site. Analysis of thissequence revealed that the unique HincII site (FIG. 21, underlined) wassituated within an ORF encoding a polypeptide of 90 amino acids. The ORFbegins with an ATG at position 1531 and terminates at position 898(positions marked by arrows in FIG. 21).

The arms for the de-ORFed insertion plasmid were derived by PCR usingpRW731.13 as template. A 596 bp arm (designated as HB) corresponding tothe region upstream from the ORF was amplified with oligonucleotidesF73PH2 (SEQ ID NO:76) (5'-GACAATCTAAGTCCTATATTAGAC-3') and F73PB (SEQ IDNO:77) (5'-GGATTTTTAGGTAGACAC-3'). A 270 bp arm (designated as EH)corresponding to the region downstream from the ORF was amplified usingoligonucleotides F75PE (SEQ ID NO:78) (5'-TCATCGTCTTCATCATCG-3') andF73PH1 (SEQ ID NO:79) (5'-GTCTTAAACTTATTGTAAGGGTATACCTG-3').

Fragment EH was digested with EcoRV to generate a 126 bp fragment. TheEcoRV site is at the 3'-end and the 5'-end was formed, by PCR, tocontain the 3' end of a HincII site. This fragment was inserted intopBS-SK (Stratagene, La Jolla, Calif.) digested with HincII to formplasmid pF7D1. The sequence was confirmed by dideoxynucleotide sequenceanalysis. The plasmid pF7D1 was linearized with ApaI, blunt-ended usingT4 DNA polymerase, and ligated to the 596 bp HB fragment. The resultantplasmid was designated as pF7D2. The entire sequence and orientationwere confirmed by nucleotide sequence analysis.

The plasmid pF7D2 was digested with EcoRV and BglII to generate a 600 bpfragment. This fragment was inserted into pBS-SK that was digested withApaI, blunt-ended with T4 DNA polymerase, and subsequently digested withBamHI. The resultant plasmid was designated as pF7D3. This plasmidcontains an HB arm of 404 bp and a EH arm of 126 bp.

The plasmid pF7D3 was linearized with XhoI and blunt-ended with theKlenow fragment of the E. coli DNA polymerase in the presence of 2 mMdNTPs. This linearized plasmid was ligated with annealedoligonucleotides F7MCSB (SEQ ID NO:80)(5'-AACGATTAGTTAGTTACTAAAAGCTTGCTGCAGCCCGGGTTTTTTATTAGTTTAGTTAGTC-3')and F7MCSA (SEQ ID NO:81)(5'-GACTAACTAACTAATAAAAAACCCGGGCTGCAGCAAGCTTTTTGTAACTAACTAATCGTT-3').This was performed to insert a multiple cloning region containing therestriction sites for HindIII, PstI and SmaI between the EH and HB arms.The resultant plasmid was designated as pF7DO.

Construction of Insertion Plasmid for the H4 Hemagglutinin at the F8Locus.

A cDNA copy encoding the avian influenza H4 derived from A/Ty/Min/833/80was obtained from Dr. R. Webster in plasmid pTM4H833. The plasmid wasdigested with HindIII and NruI and blunt-ended using the Klenow fragmentof DNA polymerase in the presence of dNTPs. The blunt-ended 2.5 kbpHindIII-NruI fragment containing the H4 coding region was inserted intothe HincII site of pIBI25 (International Biotechnologies, Inc., NewHaven, Conn.). The resulting plasmid pRW828 was partially cut withBanII, the linear product isolated and recut with HindIII. PlasmidpRW828 now with a 100 bp HindIII-BanII deletion was used as a vector forthe synthetic oligonucleotides RW152 (SEQ ID NO:82) and RW153 (SEQ IDNO:83). These oligonucleotides represent the 3' portion of the H6promoter from the EcoRV site and align the ATG of the promoter with theATG of the H4 cDNA.

    RW152 (SEQ ID NO:82):                                                                      5' GCACGGAACAAAGCTTATCGCGATATCCGTTA                                           AGTTTGTATCGTAATGCTATCAATCACGATTCTGTTCCTGTTCCTGCTCATAGC                        AGAGGGCTCATCTCAGAAT 3'                                           RW153 (SEQ ID NO:83):                                                                      5' ATTCTGAGATGAGCCCTCTGCTATGAGCAGGA                                           ACAGAATCGTGATTGATAGCATTACGATACAAACTTAACGGATATCGC                              GATAAGCTTTGTTCCGTGC 3'                                       

The oligonucleotides were annealed, cut with BanII and HindIII andinserted into the HindIII-BanII deleted pRW828 vector described above.The resulting plasmid pRW844 was cut with EcoRV and DraI and the 1.7 kbpfragment containing the 3' H6 promoted H4 coding sequence was insertedbetween the EcoRV and HincII sites of pRW846 (described previously)forming plasmid pRW848. Plasmid pRW848 therefore contains the H4 codingsequence linked to the vaccinia virus H6 promoter in the de-ORFed F8locus of fowlpox virus.

Construction of Insertion Plasmid for H5 Hemagglutinin at the F8 Locus.

A cDNA clone of avian influenza H5 derived from A/Turkey/Ireland/1378/83was received in plasmid pTH29 from Dr. R. Webster. Syntheticoligonucleotides RW10 (SEQ ID NO:84) through RW13 (SEQ ID NO:87) weredesigned to overlap the translation initiation codon of the previouslydescribed vaccinia virus H6 promoter with the ATG of the H5 gene. Thesequence continues through the 5' SalI site of the H5 gene and beginsagain at the 3' H5 DraI site containing the H5 stop codon.

    RW10 (SEQ ID NO:84):                                                                       5' GAAAAATTTAAAGTCGACCTGTTTTGTTGAGT                                           TGTTTGCGTGGTAACCAATGCAAATCTGGTC                                               ACT 3'                                                           RW11 (SEQ ID NO:85):                                                                       5' TCTAGCAAGACTGACTATTGCAAAAAGAAGCA                                           CTATTTCCTCCATTACGATACAAACTTAACG                                               GAT 3'                                                           R12 (SEQ ID NO:86):                                                                        5' ATCCGTTAAGTTTGTATCGTAATGGAGGAAA                                            TAGTGCTTCTTTTTGCAATAGTCAGTCTTGCTAGAAGTGACCAGATTT                              GCATTGGT 3'                                                      R13 (SEQ ID NO:87):                                                                        5' TACCACGCAAACAACTCAACAAAACAGGTCG                                            ACTTTAAATTTTTCTGCA 3'                                        

The oligonucleotides were annealed at 95° C. for three minutes followedby slow cooling at room temperature. This results in the followingdouble strand structure with the indicated ends. ##STR1##

Cloning of oligonucleotides between the EcoRV and PstI sites of pRW742Bresulted in pRW744. Plasmid pRW742B contains the vaccinia virus H6promoter linked to a non-pertinent gene inserted at the HincII site ofpRW731.15 described previously. Digestion with PstI and EcoRV eliminatesthe non-pertinent gene and the 3'-end of the H6 promoter. Plasmid pRW744now contains the 3' portion of the H6 promoter overlapping the ATG ofavian influenza H5. The plasmid also contains the H5 sequence throughthe 5' SalI site and the 3' sequence from the H5 stop codon (containinga DraI site). Use of the DraI site removes the H5 3' non-coding end. Theoligonucleotides add a transcription termination signal recognized byearly vaccinia virus RNA polymerase (Yuen et al., 1987). To complete theH6 promoted H5 construct, the H5 coding region was isolated as a 1.6 kpbSalI-DraI fragment from pTH29. Plasmid pRW744 was partially digestedwith DraI, the linear fragment isolated, recut with SalI and the plasmidnow with eight bases deleted between SalI and DraI was used as a vectorfor the 1.6 kpb pTH29 SalI and DraI fragment. The resulting plasmidpRW759 was cut with EcoRV and DraI. The 1.7 kbp PRW759 EcoRV-DraIfragment containing the 3' H6 promoter and the H5 gene was insertedbetween the EcoRV and HincII sites of pRW846 (previously described). Theresulting plasmid pRW849 contains the H6 promoted avian influenza virusH5 gene in the de-ORFed F8 locus.

Construction of Insertion Vector for H7 Hemagglutinin at the F7 Locus.

Plasmid pCVH71 containing the H7 hemagglutinin from A/CK/VIC/1/85 wasreceived from Dr. R. Webster. An EcoRI-BamHI fragment containing the H7gene was blunt-ended with the Klenow fragment of DNA polymerase andinserted into the HincII site of pIBI25 as PRW827. Syntheticoligonucleotides RW165 (SEQ ID NO:88) and RW166 (SEQ ID NO:89) wereannealed, cut with HincII and StyI and inserted between the EcoRV andSTyI sites of pRW827 to generate pRW845.

    RW165 (SEQ ID NO:88):                                                                      5' GTACAGGTCGACAAGCTTCCCGGGTATCGCG                                            ATATCCGTTAAGTTTGTATCGTAATGAATACTCAAATTCTAATACTCA                              CTCTTGTGGCAGCCATTCACACAAATGCAGACAAAATCTGCCTTGGAC                              ATCAT 3'                                                         RW166 (SEQ ID NO:89):                                                                      5' ATGATGTCCAAGGCAGATTTTGTCTGCATTTG                                           TGTGAATGGCTGCCACAAGAGTGAGTATTAGAATTTGAGTATTCATTA                              CGATACAAACTTAACGGATATCGCGATACCCGGGAAGCTTGTCGACCT                              GTAC 3'                                                      

Oligonucleotides RW165 (SEQ ID NO:88) and RW166 (SEQ ID NO:89) link the3' portion of the H6 promoter to the H7 gene. The 3' non-coding end ofthe H7 gene was removed by isolating the linear product of an ApaLIdigestion of pRW845, recutting it with EcoRI, isolating the largestfragment and annealing with synthetic oligonucleotides RW227 (SEQ IDNO:90) and RW228 (SEQ ID NO:91). The resulting plasmid was pRW854.

    RW227                                                                              (SEQ ID NO:90):                                                                             5' ATAACATGCGGTGCACCATTTGTATAT                                                AAGTTAACGAATTCCAAGTCAAGC 3'                                RW228                                                                              (SEQ ID NO:91):                                                                              5' GCTTGACTTGGAATTCGTTAACTTATA                                               TACAAATGGTGCACCGCATGTTAT 3'                            

The stop codon of H7 in PRW854 is followed by an HpaI site. Theintermediate H6 promoted H7 construct in the de-ORFed F7 locus(described below) was generated by moving the pRW854 EcoRV-HpaI fragmentinto pRW858 which had been cut with EcoRV and blunt-ended at its PstIsite. Plasmid pRW858 (described below) contains the H6 promoter in an F7de-ORFed insertion plasmid.

The plasmid pRW858 was constructed by insertion of an 850 bp SmaI/HpaIfragment, containing the H6 promoter linked to a non-pertinent gene,into the SmaI site of pF7DO described previously. The non-pertinentsequences were excised by digestion of pRW858 with EcoRV (site 24 bpupstream of the 3'-end of the H6 promoter) and PstI. The 3.5 kbresultant fragment was isolated and blunt-ended using the Klenowfragment of the E. coli DNA polymerase in the presence of 2 mM dNTPs.This blunt-ended fragment was ligated to a 1700 bp EcoRV/HpaI fragmentderived from pRW854 (described previously). This EcoRV/HpaI fragmentcontains the entire AIV HA (H7) gene juxtaposed 3' to the 3'-most 24 bpof the VV H6 promoter. The resultant plasmid was designated pRW861.

The 126 bp EH arm (defined previously) was lengthened in pRW861 toincrease the recombination frequency with genomic TROVAC DNA. Toaccomplish this, a 575 bp AccI/SnaBI fragment was derived from pRW731.13 (defined previously). The fragment was isolated and insertedbetween the AccI and NaeI sites of pRW861. The resultant plasmid,containing an EH arm of 725 bp and a HB arm of 404 bp flanking the AIVH7 gene, was designated as pRW869. Plasmid pRW869 therefore consists ofthe H7 coding sequence linked at its 5' end to the vaccinia virus H6promoter. The left flanking arm consists of 404 bp of TROVAC sequenceand the right flanking arm of 725 bp of TROVAC sequence which directsinsertion to the de-ORFed F7 locus.

Development of TROVAC-Avian Influenza Virus Recombinants.

Insertion plasmids containing the avian influenza virus HA codingsequences were individually transfected into TROVAC infected primary CEFcells by using the calcium phosphate precipitation method previouslydescribed (Panicali et al., 1982; Piccini et al., 1987). Positiveplaques were selected on the basis of hybridization to HA specificradiolabelled probes and subjected to sequential rounds of plaquepurification until a pure population was achieved. One representativeplaque was then amplified to produce a stock virus. Plasmid pRW849 wasused in an in vitro recombination test to produce recombinantTROVAC-AIH5 (vFP89) expressing the H5 hemagglutinin. Plasmid pRW848 wasused to produce recombinant TROVAC-AIH4 (vFP92) expressing the H4hemagglutinin. Plasmid pRW869 was used to produce recombinantTROVAC-AIH7 (vFP100) expressing the H7 hemagglutinin.

Immunofluorescence.

In influenza virus infected cells, the HA molecule is synthesized andglycosylated as a precursor molecule at the rough endoplasmic reticulum.During passage to the plasma membrane it undergoes extensivepost-translational modification culminating in proteolytic cleavage intothe disulphide linked HA₁ and HA₂ subunits and insertion into the hostcell membrane where it is subsequently incorporated into mature viralenvelopes. To determine whether the HA molecules produced in cellsinfected with the TROVAC-AIV recombinant viruses were expressed on thecell surface, immunofluorescence studies were performed. Indirectimmunofluorescence was performed as described (Taylor et al., 1990).Surface expression of the H5 hemagglutinin in TROVAC-AIH5, H4hemagglutinin in TROVAC-AIH4 and H7 hemagglutinin in TROVAC-AIH7 wasconfirmed by indirect immunofluorescence. Expression of the H5hemagglutinin was detected using a pool of monoclonal antibodiesspecific for the H5HA. Expression of the H4HA was analyzed using a goatmonospecific anti-H4 serum. Expression of the H7HA was analyzed using aH7 specific monoclonal antibody preparation.

Immunoprecipitation. It has been determined that the sequence at andaround the cleavage site of the hemagglutinin molecule plays animportant role in determining viral virulence since cleavage of thehemagglutinin polypeptide is necessary for virus particles to beinfectious. The hemagglutinin proteins of the virulent H5 and H7 virusespossess more than one basic amino acid at the carboxy terminus of HA1.It is thought that this allows cellular proteases which recognize aseries of basic amino acids to cleave the hemagglutinin and allow theinfectious virus to spread both in vitro and in vivo. The hemagglutininmolecules of H4 avirulent strains are not cleaved in tissue cultureunless exogenous trypsin is added.

In order to determine that the hemagglutinin molecules expressed by theTROVAC recombinants were authentically processed, immunoprecipitationexperiments were performed as described (Taylor et al., 1990) using thespecific reagents described above.

Immunoprecipitation analysis of the H5 hemagglutinin expressed byTROVAC-AIH5 (vFP89) showed that the glycoprotein is evident as the twocleavage products HA₁ and HA₂ with approximate molecular weights of 44and 23 kDa, respectively. No such proteins were precipitated fromuninfected cells or cells infected with parental TROVAC. Similarlyimmunoprecipitation analysis of the hemagglutinin expressed byTROVAC-AIH7 (vFP100) showed specific precipitation of the HA₂ cleavageproduct. The HA₁ cleavage product was not recognized. No proteins werespecifically precipitated from uninfected CEF cells or TROVAC infectedCEF cells. In contrast, immunoprecipitation analysis of the expressionproduct of TROVAC-AIH4 (vFP92) showed expression of only the precursorprotein HA₀. This is in agreement with the lack of cleavage of thehemagglutinins of avirulent subtypes in tissue culture. No H4 specificproteins were detected in uninfected CEF cells or cells infected withTROVAC. Generation of recombinant virus by recombination, in situhybridization of nitrocellulose filters and screening forB-galactosidase activity are as previously described (Panicali et al.,1982; Perkus et al., 1989).

Example 26 CHV gB, gC and gD Nucleotides in Vector System, ExpressionTherefrom and Use of Vector System and Expression Product

Expression of the CHV gB glycoprotein is accomplished by putting the CHVgB homolog gene under the control of the vaccinia virus I3L promoter.Expression of the CHV gc glycoprotein is accomplished by putting the CHVgC homolog gene under the control of the vaccinia virus H6 promoter.Expression of the CHV gD glycoprotein is accomplished by putting the CHVgD homolog gene under the control of the entomopox virus 42K genepromoter. The gB and gC coding is in the ATI locus and, the gD coding isin the HA locus.

Generation of Donor Plasmid.

The CHV gB coding sequence is PCR-derived. The CHV gB fragment is fusedto a PCR-derived fragment containing the I3L promoter element in aplasmid containing the cassette I3L-CHV gB in the ATI deorfed locus. TheCHV gC coding is PCR-derived and is fused in the HA deorfed locus in aplasmid.

A donor plasmid is used to insert the I3L-CHV gB--H6-CHV gC doubleconstruction in the NYVAC ATI deorfed locus.

In vitro recombination is performed on Vero cells using the donorplasmid and vP866 (NYVAC) as the rescuing virus. Standard protocols wereused to identify and purify the recombinant virus (Piccini et al.,1987). The NYVAC-based recombinant containing the CHV gB and gC genes inthe ATI deorfed locus is designated NYVAC-CHVgBgC.

Generation of Donor Plasmid.

The CHV gD coding sequence is fused to the 42K promoter and a resultingplasmid therefrom generated for insertion with the NYVAC HA deorfedlocus.

In vitro recombination is performed on Vero cells using the CHV gD-42Kdonor plasmid and recombinant vaccinia virus NYVAC-CHVgBgC (NYVACbackground) as the rescuing virus. This is performed with standardprocedures (Piccini et al., 1987). The NYVAC-based recombinantcontaining the CHV gB and gC genes in the ATI deorfed locus and the CHVgD gene in the HA deorfed locus is designated NYVAC-CHVgBgCgD.

Generation of ALVAC donor plasmid.

A plasmid donor plasmid to insert the I3L-CHV gB--H6-CHV gC--42K-CHV gDtriple construction in the ALVAC C3 deorfed locus is constructed fromthe above plasmids.

In vitro recombination is performed on primary chick embryo fibroblastsusing the donor plasmid and CPpp (ALVAC) as the rescuing virus. Standardprocedures are followed to identify and purify the generated recombinant(Piccini et al., 1987). The ALVAC-based recombinant contains the CHV gB,gC and gD genes in the C3 deorfed locus and is designatedALVAC-CHVgBgCgD.

Analysis confirms expression of the glycoproteins by the recombinantsand, the glycoproteins are substantially within the predicted sequences.

Example 27 Generation of vCP320; an Alvac Recombinant Expressing CHV gBEXPRESSING CHV gB

vCP320, an ALVAC recombinant expressing CHV gB, was generated by thefollowing procedure. A 6 kb XbaI fragment, containing the CHV gB gene,was isolated from genomic CHV DNA and cloned into the XbaI site ofpBSK+. The plasmid generated by this manipulation is called pCHV2.

The CHV gB gene was then cloned between canarypox flanking arms. Thiswas accomplished by cloning the 3,700 bp SacI-EcoRV fragment of pCHV2,containing the CHV gB gene, into the 5,800 bp SacI-NaeI fragment ofpBHVC16. (pBHVC16 contains a copy of the BHV1 gC gene cloned between C5flanking arms.) The plasmid generated by this manipulation is calledpCHV14.

Extraneous 3'-noncoding sequence was then eliminated. This wasaccomplished by cloning a 210 bp SmaI-ClaI-digested PCR fragment,containing the 3'-end of the gB gene, into the 5,500 bp partialSmaI-ClaI fragment of pCHV14. (This PCR fragment was generated from theplasmid, pCHV2, with the primers, CHVP39 (SEQ ID NO: 92;5'-TAAGAATGGTAATTCT-3') and CHVP40 (SEQ ID NO: 93;5'-TTCCCGGGTTAAACTTTACTTTCATTTTC-3').) The plasmid generated by thismanipulation is called pCHV15.

The I3L promoter was then cloned upstream from the gB initiation codon.In addition, 3 T₅ NT early transcription termination signal sequenceslocated in the 5'-end of the gB gene were modified. This wasaccomplished by cloning a 140 bp ScaI-SalI-digested PCR fragment,containing the I3L promoter and the T₅ NT-modified 5'-end of the gBgene, into the 6,300 bp ScaI-SalI fragment of pCHV15. (This PCR fragmentwas generated from the plasmid, pCHV2, with the primers,

    CHVP42 (SEQ ID NO: 94;                                                                      5'-TTGTCGACTGAGATAAAGTGAAAATATATCATTATATTACAAAGTACAATTATTTAG                  G                                                                             TTTAATCATGTTTTCATTGTATCTATAT-3')                                and                                                                           CHVP78 (SEQ ID NO: 95;                                                                      5'-TTAGTACTTTCCGGTGTTGTTGGATCACATATTATTAAAGTATAAATAATAAAGAA-                  3').)                                                       

The plasmid generated by this manipulation is called pCHV27.

An error in the sequence flanking the ScaI-SalI fragment of pCHV27 wasthen corrected. This was accomplished by cloning the 180 bp ScaI-SalIfragment of pCHV27, containing the I3L promoter and the T₅ NT-modified5'-end of the gB gene, into the 6,300 bp ScaI-SalI fragment of pCHV15.The plasmid generated by this manipulation is called pCHV28.

An early transcription termination signal sequence near the 3'-end ofthe CHV gB gene was then modified. This was accomplished by cloning a330 bp SpeI-Asp718-digested PCR fragment, containing the T₅ NT-modifiedregion of the CHV gB gene, into the 5,450 bp SpeI-Asp718 fragment ofpCHV28. (This PCR fragment was generated from a 150 bp PCR fragment, a280 bp PCR fragment and the primers, CHVP89 (SEQ ID NO: 96;5'-TGGAATGAAGTTATGAAACT-3') and CHVP92 (SEQ ID NO: 97;5'-TGCACTGATCATTTCAATTTC-3'). The 150 bp PCR fragment was generated fromthe plasmid, pCHV2, with the primers, CHVP89 (SEQ ID NO: 96;5'-TGGAATGAAGTTATGAAACT-3') and CHVP90 (SEQ IS NO: 98;5'-TGGAATTTTGAATGAAAACACTAGAACC-3'). The 280 bp PCR fragment wasgenerated from the plasmid, pCHV2, with the primers, CHVP91 (SEQ ID NO:99; 5'-TTCTAGTGTTTTCATTCAAAATTCCAT-3') and CHVP92 (SEQ ID NO: 97;5'-TGCACTGATCATTTCAATTTC-3').) The plasmid generated by thismanipulation is called pCHV31.

An early transcription termination signal sequence in the middle of theCHV gB gene was then modified. This was accomplished by cloning a 480 bpBamHI-BsaBI-digested PCR fragment, containing the T₅ NT-modified regionof the gB gene, into the 5,000 bp BamHI-BsaBI fragment of pCHV31. (ThisPCR fragment was generated from a 380 bp PCR fragment, a 210 bp PCRfragment and the primers, CHVP87 (SEQ ID NO: 100;5'-CCTTCAAAGTTTAATACACC-3') and CHVP94 (SEQ ID NO: 101;5'-TATGGCTTCACGTTTGGCAC-3'). The 380 bp PCR fragment was generated fromthe plasmid, pCHV2, with the primers, CHVP93 (SEQ ID NO: 102;5'-CACCGGGGATATAATTCATATGTCCCCTTTCTTTGGATTACGAGATGGT-3') and CHVP94 (SEQID NO: 101; 5'-TATGGCTTCACGTTTGGCAC-3'). The 210 bp PCR fragment wasgenerated from the plasmid, pCHV2, with the primers, CHVP87 (SEQ ID NO:100; 5'-CCTTCAAAGTTTAATACACC-3') and CHVP88 (SEQ ID NO: 103;5'-CCATCTCGTAATCCAAAGAAAGGGGACATATGAAT-3').) The plasmid generated bythis manipulation is called pCHV32.

A portion of the gB gene removed in the previous manipulation was thencloned back into pCHV32. This was accomplished by cloning the 2,000 bppartial BsaBI-PstI fragment of pCHV31, containing the 3'-end of the gBgene removed in the previous manipulation, into the 5,450 bp BsaBI-PstIfragment of pCHV32. The plasmid generated by this manipulation is calledpCHV36.

The I3L-promoted gB gene was then cloned between C6 flanking arms. Thiswas accomplished by cloning the 2,750 bp SalI-SmaI fragment of pCHV36,containing the I3L-promoted gB gene, into the 4,350 bp SalI-SmaIfragment of pHIV34. (pHIV34 contains a copy of the H6-promoted HIV2gp120 (+TM) gene cloned between C6 flanking arms.) The plasmid generatedby this manipulation is called pCHV37. The DNA sequence of theI3L-promoted gB gene in pCHV37 (SEQ ID NOS:104, 105, 106) is shown inFIG. 23. The DNA sequence of the ALVAC C6 flanking arms (SEQ ID NOS:107, 108) is shown in FIG. 24.

pCHV37 was used in in vitro recombination experiments with ALVAC as therescuing virus to yield vCP320.

Immunoprecipitation analysis was performed to determine whether vCP320expresses CHV gB. MDCK cell monolayers were either mock infected orinfected with the parental virus (ALVAC) (m.o.i.=15 PFU/cell), vCP320(m.o.i.=15 PFU/cell) or CHV (m.o.i.=10 PFU/cell). Following an houradsorption period, the inoculum was removed and the cells were overlayedwith 2 mls of modified Eagle's medium (minus cysteine) containing 2%dialyzed fetal bovine serum and [³⁵ S]-cysteine (50 μCi/ml). The lysateswere harvested at 18 hrs post-infection in 1 ml 3×buffer A (450 mM NaCl,3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide and 0.6 mg/mlPMSF) and analyzed for CHV gB expression using a 1:100 dilution of agB-specific monoclonal antibody, 1125B2 (obtained from Dr. MichelRiviere, Rhone Merieux, Lyon, France). Lysates, precleared with normalmouse sera and a goat anti-mouse-protein A-sepharose complex, wereincubated overnight at 4° C. with a monoclonal antibody-goatanti-mouse-protein A-sepharose complex and washed 4× with 1×buffer A and2× with a LiCl₂ /urea buffer. Precipitated proteins were dissociatedfrom the immune complexes by the addition of 2×Laemmli's buffer (125 mMTris (pH=6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boilingfor 5 min. Proteins were fractionated on an SDS-polyacrylamide gel,fixed and treated with 1 M Na-salicylate for fluorography. Proteins ofthe appropriate size were precipitated from CHV-infected cells (lane D)and vCP320-infected cells (lane C), but were not precipitated frommock-infected cells (lane A) or ALVAC-infected cells (lane B) (FIG. 25).These results indicate that vCP320 expresses CHV gB.

Example 28 Generation of vCP322; an Alvac Recombinant Expressing CHV gCEXPRESSING CHV gC

vCP322, an ALVAC recombinant expressing CHV gC, was generated by thefollowing procedure. A 2.2 kb EcoRI fragment, containing the CHV gCgene, was isolated from genomic CHV DNA and cloned into the EcoRI siteof pVQH6CP3LSA. (pVQH6CP3LSA contains a copy of the H6 promoter clonedbetween C3 flanking arms.) This manipulation positions the gC genedownstream from the H6 promoter and between C3 flanking arms. Theplasmid generated by this manipulation is called pCHV17.

Extraneous 3'-noncoding sequence was then eliminated and 3 T₅ NT earlytranscription termination signal sequences located near the 3'-end ofthe gC gene were modified. This was accomplished by cloning theoligonucleotides,

    CHVL66 (SEQ ID NO: 109:                                                                     5'-CGATGTTAATAAGTATTACCACAATAATTGGTGGAGCCATTTTCGTTATAGTATTGA                  TTT                                                                           TCATAACAGCTTTATGTTTCTATTGTTCAAAAAATAATAAGATCTAACTGCA-3')        and                                                                           CHVL67 (SEQ ID NO: 110;                                                                     5'-GTTAGATCTTATTATTTTTTGAACAATAGAAACATAAAGCTGTTATGAAAATCAATA                  CTA                                                                           TAACGAAAATGGCTCCACCAATTATTGTGGTAATACTTATTAACAT-3'),         

into the 8,400 bp partial ClaI-PstI fragment of pCHV17. The plasmidgenerated by this manipulation is called pCHV20.

The initiation codon of the gC gene was then aligned with the initiationcodon of the H6 promoter. In addition, 2 early transcription terminationsignal sequences were modified. This was accomplished by cloning a 740bp NruI-BsrGI-digested PCR fragment, containing the 3'-end of the H6promoter and the 5'-end of the T₅ NT-modified gC gene, into the 7,900 bpNruI-BsrGI fragment of pCHV20. (This PCR fragment was generated from a500 bp PCR fragment, a 300 bp PCR fragment and the oligonucleotides,CHVP96 (SEQ ID NO: 111; 5'-CGTAGATTCCAATGGAAAGT-3') and CHVP97 (SEQ IDNO: 112; 5'-TTTTCGCGATATCCGTTAAGT-3'). The 500 bp PCR fragment wasgenerated from the plasmid, pCHV13, with the oligonucleotides, CHVP68(SEQ ID NO: 113;5'-TTTTCGCGATATCCGTTAAGTTTGTATCGTAATGAGTTTTAAAAATTTCTATCTAATATATGTAATTATAATTTTCATAAACTCGATAATAAC-3') and CHVP69 (SEQ ID NO: 114;5'-TTTGTATACCTAATAAGAAATCATTATAAAAGT-3'). The 300 bp PCR fragment wasgenerated from the plasmid, pCHV13, with the oligonucleotides, CHVP95(SEQ ID NO: 115; 5'-CTTTTATAATGATTTCTTATTAGGTATACAAAATC-3') and CHVP96(SEQ ID NO: 111; 5'-CGTAGATTCCAATGGAAAGT-3'). pCHV13 was obtained bycloning the 2.2 kb EcoRI CHV genomic fragment, containing the gC gene,into the EcoRI site of pBSK+.) The plasmid generated by thismanipulation is called pCHV38.

The H6-promoted gC gene was then cloned between C6 flanking arms. Thiswas accomplished by cloning the 1,400 bp NruI-PstI fragment of pCHV38,containing the H6-promoted gC gene, and the oligonucleotide, CHVL98 (SEQID NO:116; 5'-AATTTGCA-3'), into the 4,500 bp NruI-EcoRI fragment ofpHIV34. (pHIV34 contains a copy of the H6-promoted HIV2 gp120 (+TM) genecloned between C6 flanking arms.) The plasmid generated by thismanipulation is called pCHV40. The DNA sequence of the H6-promoted gCgene in pCHV40 (SEQ ID NOS: 117, 118, 119) is shown in FIG. 26. The DNAsequence of the ALVAC C6 flanking arms (SEQ ID NOS: 107, 108) is shownin FIG. 24.

pCHV40 was used in in vitro recombination experiments with ALVAC as therescuing virus to yield vCP322.

Immunoprecipitation analysis was performed to determine whether vCP322expresses CHV gC. MDCK cell monolayers were either mock infected orinfected with the parental virus (ALVAC) (m.o.i.=15 PFU/cell), vCP322(m.o.i.=15 PFU/cell) or CHV (m.o.i.=10 PFU/cell). Following an houradsorption period, the inoculum was removed and the cells were overlayedwith 2 mls of modified Eagle's medium (minus cysteine) containing 2%dialyzed fetal bovine serum and [³⁵ S]-cysteine (50 μCi/ml). The lysateswere harvested at 18 hrs post-infection in 1 ml 3×buffer A (450 mM NaCl,3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide and 0.6 mg/mlPMSF) and analyzed for CHV gC expression using a 1:100 dilution of agC-specific monoclonal antibody, 2011A9 (obtained from Dr. MichelRiviere, Rhone Merieux, Lyon, France). Lysates, precleared with normalmouse sera and a goat anti-mouse-protein A-sepharose complex, wereincubated overnight at 4° C. with a monoclonal antibody-goatanti-mouse-protein A-sepharose complex and washed 4× with 1×buffer A and2× with a LiCl₂ /urea buffer. Precipitated proteins were dissociatedfrom the immune complexes by the addition of 2×Laemmli's buffer (125 mMTris (pH=6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boilingfor 5 min. Proteins were fractionated on an SDS-polyacrylamide gel,fixed and treated with 1 M Na-salicylate for fluorography. Proteins ofthe appropriate size were precipitated from CHV-infected cells (lane D)and vCP322-infected cells (lane C), but were not precipitated frommock-infected cells (lane A) or ALVAC-infected cells (lane B) (FIG. 27).These results indicate that vCP322 expresses CHV gC.

Example 29 Generation of VCP294; An Alvac Recombinant Expressing CHV gD

vCP294, an ALVAC recombinant expressing CHV gD, was generated by thefollowing procedure. A 7 kb PstI fragment, containing the CHV gD gene,was isolated from genomic CHV DNA and cloned into the PstI site ofpBSK+. The plasmid generated by this manipulation is called pCHV11.

The CHV gD gene was then cloned between canarypox flanking arms. Thiswas accomplished by cloning the 1,475 bp PstI-SnaBI fragment of pCHV11,containing the CHV gD gene, into the 5,600 bp PstI-SmaI fragment ofpHIV34. (pHIV34 contains a copy of the H6-promoted HIV2 gp120 (+TM) genecloned between C6 flanking arms.) This places the CHV gD gene between C6flanking arms and downstream from the H6 promoter. The plasmid generatedby this manipulation is called pCHV18.

The initiation codon of the H6 promoter was then aligned with theinitiation codon of the CHV gD gene. This was accomplished by cloningthe oligonucleotides,

    CHVL81 (SEQ ID NO: 120;                                                                     5'-CGATATCCGTTAAGTTTGTATCGTAATGATTAAACTTCTATTTATCTTATTTTATTT                  TAA                                                                           CCCAATAA-3')                                                    and                                                                           CHVL82 (SEQ ID NO: 121;                                                                     5'-TTGGGTTAAAATAAAATAAGATAAATAGAAGTTTAATCATTACGATACAAACTTAAC                  GGA                                                                           TATCG-3'),                                                  

into the 5,600 bp NruI-PflMI fragment of pCHV18. The plasmid generatedby this manipulation is called pCHV21.

Three early transcription termination signal sequences at the 3'-end ofthe CHVgD gene were then modified. This was accomplished by cloning the1,400 bp BglII-Asp718 fragment of pCHV22, containing the "T₅NT-modified" 3'-end of the CHV gD gene and C3 flanking arm, into the3,700 bp BglII-Asp718 fragment of pCHV21. (pCHV22 was generated bycloning a 430 bp BglII-EcoRI-digested PCR fragment, containing the "T₅NT-modified" 3'-end of the CHV gD gene, into the 3,900 bp BglII-EcoRIfragment of pHIV43 (pHIV43 contains a copy of the H6-promoted HIV1gp120-murine IL-2 fusion gene cloned between C3 flanking arms). (ThisPCR fragment was generated from the plasmid, pCHV18, with theoligonucleotides,

    CHVP79 (SEQ ID NO: 122;                                                                     5'-TTGAATTCCTAAACATTTGTTGTTAATTTTTTATAATTAATTATATATTTTTTGTC                   TTTTATAAACAAAGAAT-3')                                           and                                                                           CHVP80 (SEQ ID NO:123;                                                                      5'-TTAGATCTGTAGGAGCATCAAAAGTTGACGATGAACTTTTGTATCTAAATAGAGC                    TGGTCCCCAAACCCTGCTTAAATATTATGTTATTAAAGATTTCTAT-3').))       

The plasmid generated by this manipulation is called pCHV24.

The "T₅ NT-modified" central region of the CHV gD gene was then clonedinto pCHV24. This was accomplished by cloning a 240 bp BglII-digestedPCR fragment, containing the "T₅ NT-modified" central region of the CHVgD gene, into the BglII site of pCHV24. (This PCR fragment was generatedfrom the plasmid, pCHV18, with the oligonucleotides,

    CHVP83 (SEQ ID NO: 124;                                                                     5'-TTAGATCTAGATTCCTTACACCATTCCATAAAAGTTGGTTCAAATTTATCTTCTTTA                  GAG                                                                           AAATAACAAGTTTCTCGTGGTAATTGAACCATAAAATCAGTATAGAAAAC-3')          and                                                                           CHVP84 (SEQ ID NO: 125;                                                                     5'-TATTTTGATTGTGATCC-3').)                                  

The plasmid generated by this manipulation is called pCHV25.

The C3 flanking arm was then replaced with the C6 flanking arm. This wasaccomplished by cloning the 1,160 bp EcoRI-Asp718 fragment of pCHV21,containing the C6 flanking arm, into the 4,100 bp EcoRI-Asp718 fragmentof pCHV25. The plasmid generated by this manipulation is called pCHV26.The DNA sequence of the H6-promoted gD gene in pCHV26 (SEQ ID NOS: 107,108) is shown in FIG. 28. The DNA sequence of the ALVAC C6 flanking arms(SEQ ID NOS: 107, 105) is shown in FIG. 24.

pCHV26 was used in in vitro recombination experiments with ALVAC as therescuing virus to yield vCP294.

Immunoprecipitation analysis was performed to determine whether vCP294expresses CHV gD. MDCK cell monolayers were either mock infected orinfected with the parental virus (ALVAC) (m.o.i.=15 PFU/cell), vCP294(m.o.i.=15 PFU/cell) or CHV (m.o.i.=10 PFU/cell). Following an houradsorption period, the inoculum was removed and the cells were overlayedwith 2 mls of modified Eagle's medium (minus cysteine) containing 2%dialyzed fetal bovine serum and [³⁵ S]-cysteine (50 μCi/ml). The lysateswere harvested at 18 hrs post-infection in 1 ml 3×buffer A (450 mM NaCl,3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide and 0.6 mg/mlPMSF) and analyzed for CHV gD expression using a 1:100 dilution of agD-specific monoclonal antibody, 208D11 (obtained from Dr. MichelRiviere, Rhone Merieux, Lyon, France). Lysates, precleared with normalmouse sera and a goat anti-mouse-protein A-sepharose complex, wereincubated overnight at 4° C. with a monoclonal antibody-goatanti-mouse-protein A-sepharose complex and washed 4× with 1×buffer A and2× with a LiCl₂ /urea buffer. Precipitated proteins were dissociatedfrom the immune complexes by the addition of 2×Laemmli's buffer (125 mMTris (pH=6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boilingfor 5 min. Proteins were fractionated on an SDS-polyacrylamide gel,fixed and treated with 1 M Na-salicylate for fluorography. Proteins ofsimilar size were precipitated from CHV-infected cells (lane D) andvCP294-infected cells (lane C), but were not precipitated frommock-infected cells (lane A) or ALVAC-infected cells (lane B) (FIG. 29).These results indicate that vCP294 expresses CHV gD.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

The recombinants can be used to stimulate an antibody or immune responsein pups and adult dogs against CHV and, so too can the expressionproducts which can be isolated from cells infected by the recombinants.Further, the recombinants or the expression products therefrom can beused to generate antibodies in an animal administered the recombinantsor the expression products therefrom and, the antibodies can be furtherused as described herein.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 128                                           - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3000 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - TTTTCTGGAT TTCAGCTATG TCCTTCGGGA GTTTATATAA CTTATGAAGA AA - #ACTGTCCT         60                                                                          - TTGGTAGCAG TTTTACAAAG CGGTGTAAAT TGCGAAATTG GACCAACTAC AA - #CTGTAATA        120                                                                          - TACGACAGTG ATATTTTTTC TCTTCTTTAT ACCGTTCTTC AAAAATTGGC TC - #CTGGTGTT        180                                                                          - AATATAGAAA TTTGATAAGT ATGTTTTCAT TGTATCTATA TATTTTTTTT AT - #TATTTATA        240                                                                          - CTTTAATAAT ATGTGATCCA ACAACACCGG AAAGTACTAT TAATCCATTA AA - #TCATCACA        300                                                                          - ATTTATCAAC ACCTAAACCT ACTTCGGATG ATATTCGTGA AATTTTACGT GA - #ATCCCAAA        360                                                                          - TTGAATCTGA TGATACATCA ACATTTTACA TGTGCCCACC ACCATCGGGA TC - #AACATTGG        420                                                                          - TGCGTTTGGA GCCACCTAGA GCATGTCCTA ACTATAAACT TGGTAAAAAT TT - #TACAGAAG        480                                                                          - GAATTGCTGT AATATTTAAG GAAAATATTT CTCCTTATAA ATTTAAAGCT AA - #TATATACT        540                                                                          - ACAAAAATAT TATTATCACC ACTGTATGGT CTGGAAGCAC ATATGCAGTA AT - #TACTAATA        600                                                                          - GATATACAGA TCGTGTACCT ATAGGTGTTC CTGAAATTAC AGAGTTGATT GA - #TAGAAGAG        660                                                                          - GTATGTGTTT ATCAAAAGCT GATTATATTC GTAATAATTA TGAATTTACC GC - #ATTTGATA        720                                                                          - AGGATGAAGA CCCCAGAGAA GTTCATTTAA AGCCTTCAAA GTTTAATACA CC - #AGGATCCC        780                                                                          - GTGGATGGCA TACAGTTAAT GATACTTACA CAAAAATTGG GGGTTCTGGA TT - #TTATCATT        840                                                                          - CTGGAACATC TGTAAATTGT ATAGTTGAAG AAGTTGATGC CAGATCTGTT TA - #TCCATATG        900                                                                          - ATTCATTTGC TATCTCCACC GGGGATATAA TTCATATGTC CCCTTTTTTT GG - #ATTACGAG        960                                                                          - ATGGTGCTCA TACTGAATAT ATTAGTTATT CAACTGATAG ATTTCAACAA AT - #AGAAGGTT       1020                                                                          - ATTATCCTAT CGACTTAGAT ACTAGACTAC AGCTTGGTGC ACCAGTTTCT AG - #GAATTTTT       1080                                                                          - TAACAACACA ACACGTTACT GTTGCTTGGA ATTGGGTTCC AAAAATTCGT GA - #AGTGTGTA       1140                                                                          - CTTTGGCTAA ATGGCGTGAA ATTGATGAAA TTATTCGTGA TGAGTATAAG GG - #ATCTTACA       1200                                                                          - GATTTACAGC AAAATCAATA TCTGCAACAT TTATTTCTGA TACTACTCAA TT - #TGATATTG       1260                                                                          - ATCGTGTAAA GTTAAGTGAT TGTGCCAAAC GTGAAGCCAT AGAAGCTATT GA - #TAAGATCT       1320                                                                          - ACAAAAAAAA ATATAATAAA ACTCATATTC AAACAGGAGA ATTGGAAACA TA - #CTTGGCTA       1380                                                                          - GAGGGGGATT TATTATAGCA TTTAGACCAA TGATTAGTAA TGAGTTAGCA AA - #ATTGTATA       1440                                                                          - TAAATGAGTT AGTAAGATCT AATCGTACGG TTGATTTGAA ATCTCTTTTA AA - #TCCATCTG       1500                                                                          - TAAGAGGGGG GGCTAGAAAG AGAAGATCAG TAGAGGAAAA TAAAAGATCA AA - #ACGTAATA       1560                                                                          - TTGAAGGTGG TATTGAAAAT GTAAATAATT CAACAATAAT TAAGACAACT TC - #ATCTGTTC       1620                                                                          - ATTTTGCTAT GCTTCAGTTT GCCTATGATC ATATTCAATC ACATGTTAAT GA - #AATGCTTA       1680                                                                          - GTAGAATTGC AACTGCATGG TGTAATCTTC AAAATAAAGA GAGAACCCTT TG - #GAATGAAG       1740                                                                          - TTATGAAACT TAATCCAACT AGTGTGGCTT CGGTTGCTAT GGATCAAAGA GT - #TTCAGCAC       1800                                                                          - GAATGTTAGG GGATGTTCTT GCAGTTACTC AATGTGTTAA TATATCAGGT TC - #TAGTGTTT       1860                                                                          - TTATTCAAAA TTCCATGCGT GTTTTAGGGT CAACAACTAC ATGTTACAGT CG - #TCCTCTTA       1920                                                                          - TATCATTTAA AGCACTAGAA AACTCAACTA ACTATATTGA AGGACAACTT GG - #GGAAAATA       1980                                                                          - ATGAACTATT AGTAGAACGA AAGCTAATTG AACCATGTAC AGCTAACCAT AA - #AAGATATT       2040                                                                          - TTAAATTTGG TGCAGATTAT GTATATTTTG AAAACTATGC ATATGTTCGA AA - #GGTACCTC       2100                                                                          - TTAATGAAAT TGAAATGATC AGTGCATATG TAGATCTTAA TATTACATTA CT - #TGAGGATC       2160                                                                          - GTGAATTTTT ACCACTAGAG GTATATACTC GAGCAGAGTT AGAAGATACA GG - #ACTATTGG       2220                                                                          - ACTATAGTGA GATTCAACGT AGAAATCAAC TACATGCACT TAAGTTTTAT GA - #TATTGACA       2280                                                                          - GTGTTGTAAA AGTTGATAAT AATGTTGTAA TTATGAGGGG CATTGCAAAT TT - #TTTCCAAG       2340                                                                          - GACTTGGAGA TGTTGGAGCG GGATTTGGAA AAGTTGTTTT GGGTGCTGCA AA - #TGCTGTTA       2400                                                                          - TTGCAACTGT TTCTGGAGTG TCCTCGTTTC TTAATAACCC ATTTGGGGCG CT - #AGCCGTTG       2460                                                                          - GATTGCTGAT TTTAGCTGGA CTATTTGCAG CGTTTTTGGC TTATAGATAT GT - #TTCTAAAC       2520                                                                          - TTAAGTCAAA TCCAATGAAA GCACTATACC CAGTAACTAC AAAAAATTTA AA - #AGAAAGTG       2580                                                                          - TTAAGAATGG TAATTCTGGA AATAATAGTG ATGGAGAAGA AAATGATGAT AA - #TATCGATG       2640                                                                          - AAGAAAAGCT TCAACAAGCT AAAGAAATGA TTAAATATAT GTCTCTAGTT TC - #TGCTATGG       2700                                                                          - AACAGCAGGA ACATAAAGCT ATTAAAAAAA ATAGTGGCCC TGCCCTTCTA GC - #AAGTCACA       2760                                                                          - TTACAAACCT ATCTCTTAAA CATCGTGGTC CAAAATACAA ACGTTTGAAA AA - #TGTAAATG       2820                                                                          - AAAATGAAAG TAAAGTTTAA TAAAAAATTT AAATATTACG TAAAATTTTC TG - #ACTCTGCC       2880                                                                          - CACTTTTTTT ATAATATAAA TTTTAGAAAA TTTTACTCAT TTTATTATCT TT - #TATAAACC       2940                                                                          - TCCAACTATT TATAAAGGAT AATAAATGGA CATTTCTGCG GTGCCTGTAT AT - #CCTACTAA       3000                                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 879 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Phe Ser Leu Tyr Leu Tyr Ile Phe Phe Il - #e Ile Tyr Thr Leu Ile         #                15                                                           - Ile Cys Asp Pro Thr Thr Pro Glu Ser Thr Il - #e Asn Pro Leu Asn His         #            30                                                               - His Asn Leu Ser Thr Pro Lys Pro Thr Ser As - #p Asp Ile Arg Glu Ile         #        45                                                                   - Leu Arg Glu Ser Gln Ile Glu Ser Asp Asp Th - #r Ser Thr Phe Tyr Met         #    60                                                                       - Cys Pro Pro Pro Ser Gly Ser Thr Leu Val Ar - #g Leu Glu Pro Pro Arg         #80                                                                           - Ala Cys Pro Asn Tyr Lys Leu Gly Lys Asn Ph - #e Thr Glu Gly Ile Ala         #                95                                                           - Val Ile Phe Lys Glu Asn Ile Ser Pro Tyr Ly - #s Phe Lys Ala Asn Ile         #           110                                                               - Tyr Tyr Lys Asn Ile Ile Ile Thr Thr Val Tr - #p Ser Gly Ser Thr Tyr         #       125                                                                   - Ala Val Ile Thr Asn Arg Tyr Thr Asp Arg Va - #l Pro Ile Gly Val Pro         #   140                                                                       - Glu Ile Thr Glu Leu Ile Asp Arg Arg Gly Me - #t Cys Leu Ser Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asp Tyr Ile Arg Asn Asn Tyr Glu Phe Thr Al - #a Phe Asp Lys Asp Glu         #               175                                                           - Asp Pro Arg Glu Val His Leu Lys Pro Ser Ly - #s Phe Asn Thr Pro Gly         #           190                                                               - Ser Arg Gly Trp His Thr Val Asn Asp Thr Ty - #r Thr Lys Ile Gly Gly         #       205                                                                   - Ser Gly Phe Tyr His Ser Gly Thr Ser Val As - #n Cys Ile Val Glu Glu         #   220                                                                       - Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Se - #r Phe Ala Ile Ser Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Asp Ile Ile His Met Ser Pro Phe Phe Gl - #y Leu Arg Asp Gly Ala         #               255                                                           - His Thr Glu Tyr Ile Ser Tyr Ser Thr Asp Ar - #g Phe Gln Gln Ile Glu         #           270                                                               - Gly Tyr Tyr Pro Ile Asp Leu Asp Thr Arg Le - #u Gln Leu Gly Ala Pro         #       285                                                                   - Val Ser Arg Asn Phe Leu Thr Thr Gln His Va - #l Thr Val Ala Trp Asn         #   300                                                                       - Trp Val Pro Lys Ile Arg Glu Val Cys Thr Le - #u Ala Lys Trp Arg Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Asp Glu Ile Ile Arg Asp Glu Tyr Lys Gl - #y Ser Tyr Arg Phe Thr         #               335                                                           - Ala Lys Ser Ile Ser Ala Thr Phe Ile Ser As - #p Thr Thr Gln Phe Asp         #           350                                                               - Ile Asp Arg Val Lys Leu Ser Asp Cys Ala Ly - #s Arg Glu Ala Ile Glu         #       365                                                                   - Ala Ile Asp Lys Ile Tyr Lys Lys Lys Tyr As - #n Lys Thr His Ile Gln         #   380                                                                       - Thr Gly Glu Leu Glu Thr Tyr Leu Ala Arg Gl - #y Gly Phe Ile Ile Ala         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Phe Arg Pro Met Ile Ser Asn Glu Leu Ala Ly - #s Leu Tyr Ile Asn Glu         #               415                                                           - Leu Val Arg Ser Asn Arg Thr Val Asp Leu Ly - #s Ser Leu Leu Asn Pro         #           430                                                               - Ser Val Arg Gly Gly Ala Arg Lys Arg Arg Se - #r Val Glu Glu Asn Lys         #       445                                                                   - Arg Ser Lys Arg Asn Ile Glu Gly Gly Ile Gl - #u Asn Val Asn Asn Ser         #   460                                                                       - Thr Ile Ile Lys Thr Thr Ser Ser Val His Ph - #e Ala Met Leu Gln Phe         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ala Tyr Asp His Ile Gln Ser His Val Asn Gl - #u Met Leu Ser Arg Ile         #               495                                                           - Ala Thr Ala Trp Cys Asn Leu Gln Asn Lys Gl - #u Arg Thr Leu Trp Asn         #           510                                                               - Glu Val Met Lys Leu Asn Pro Thr Ser Val Al - #a Ser Val Ala Met Asp         #       525                                                                   - Gln Arg Val Ser Ala Arg Met Leu Gly Asp Va - #l Leu Ala Val Thr Gln         #   540                                                                       - Cys Val Asn Ile Ser Gly Ser Ser Val Phe Il - #e Gln Asn Ser Met Arg         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Val Leu Gly Ser Thr Thr Thr Cys Tyr Ser Ar - #g Pro Leu Ile Ser Phe         #               575                                                           - Lys Ala Leu Glu Asn Ser Thr Asn Tyr Ile Gl - #u Gly Gln Leu Gly Glu         #           590                                                               - Asn Asn Glu Leu Leu Val Glu Arg Lys Leu Il - #e Glu Pro Cys Thr Ala         #       605                                                                   - Asn His Lys Arg Tyr Phe Lys Phe Gly Ala As - #p Tyr Val Tyr Phe Glu         #   620                                                                       - Asn Tyr Ala Tyr Val Arg Lys Val Pro Leu As - #n Glu Ile Glu Met Ile         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ser Ala Tyr Val Asp Leu Asn Ile Thr Leu Le - #u Glu Asp Arg Glu Phe         #               655                                                           - Leu Pro Leu Glu Val Tyr Thr Arg Ala Glu Le - #u Glu Asp Thr Gly Leu         #           670                                                               - Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gl - #n Leu His Ala Leu Lys         #       685                                                                   - Phe Tyr Asp Ile Asp Ser Val Val Lys Val As - #p Asn Asn Val Val Ile         #   700                                                                       - Met Arg Gly Ile Ala Asn Phe Phe Gln Gly Le - #u Gly Asp Val Gly Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Phe Gly Lys Val Val Leu Gly Ala Ala As - #n Ala Val Ile Ala Thr         #               735                                                           - Val Ser Gly Val Ser Ser Phe Leu Asn Asn Pr - #o Phe Gly Ala Leu Ala         #           750                                                               - Val Gly Leu Leu Ile Leu Ala Gly Leu Phe Al - #a Ala Phe Leu Ala Tyr         #       765                                                                   - Arg Tyr Val Ser Lys Leu Lys Ser Asn Pro Me - #t Lys Ala Leu Tyr Pro         #   780                                                                       - Val Thr Thr Lys Asn Leu Lys Glu Ser Val Ly - #s Asn Gly Asn Ser Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asn Asn Ser Asp Gly Glu Glu Asn Asp Asp As - #n Ile Asp Glu Glu Lys         #               815                                                           - Leu Gln Gln Ala Lys Glu Met Ile Lys Tyr Me - #t Ser Leu Val Ser Ala         #           830                                                               - Met Glu Gln Gln Glu His Lys Ala Ile Lys Ly - #s Asn Ser Gly Pro Ala         #       845                                                                   - Leu Leu Ala Ser His Ile Thr Asn Leu Ser Le - #u Lys His Arg Gly Pro         #   860                                                                       - Lys Tyr Lys Arg Leu Lys Asn Val Asn Glu As - #n Glu Ser Lys Val             865                 8 - #70                 8 - #75                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 879 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Phe Ser Leu Tyr Leu Tyr Ile Phe Phe Il - #e Ile Tyr Thr Leu Ile         #                15                                                           - Ile Cys Asp Pro Thr Thr Pro Glu Ser Thr Il - #e Asn Pro Leu Asn His         #            30                                                               - His Asn Leu Ser Thr Pro Lys Pro Thr Ser As - #p Asp Ile Arg Glu Ile         #        45                                                                   - Leu Arg Glu Ser Gln Ile Glu Ser Asp Asp Th - #r Ser Thr Phe Tyr Met         #    60                                                                       - Cys Pro Pro Pro Ser Gly Ser Thr Leu Val Ar - #g Leu Glu Pro Pro Arg         #80                                                                           - Ala Cys Pro Asn Tyr Lys Leu Gly Lys Asn Ph - #e Thr Glu Gly Ile Ala         #                95                                                           - Val Ile Phe Lys Glu Asn Ile Ser Pro Tyr Ly - #s Phe Lys Ala Asn Ile         #           110                                                               - Tyr Tyr Lys Asn Ile Ile Ile Thr Thr Val Tr - #p Ser Gly Ser Thr Tyr         #       125                                                                   - Ala Val Ile Thr Asn Arg Tyr Thr Asp Arg Va - #l Pro Ile Gly Val Pro         #   140                                                                       - Glu Ile Thr Glu Leu Ile Asp Arg Arg Gly Me - #t Cys Leu Ser Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asp Tyr Ile Arg Asn Asn Tyr Glu Phe Thr Al - #a Phe Asp Lys Asp Glu         #               175                                                           - Asp Pro Arg Glu Val His Leu Lys Pro Ser Ly - #s Phe Asn Thr Pro Gly         #           190                                                               - Ser Arg Gly Trp His Thr Val Asn Asp Thr Ty - #r Thr Lys Ile Gly Gly         #       205                                                                   - Ser Gly Phe Tyr His Ser Gly Thr Ser Val As - #n Cys Ile Val Glu Glu         #   220                                                                       - Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Se - #r Phe Ala Ile Ser Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Asp Ile Ile His Met Ser Pro Phe Phe Gl - #y Leu Arg Asp Gly Ala         #               255                                                           - His Thr Glu Tyr Ile Ser Tyr Ser Thr Asp Ar - #g Phe Gln Gln Ile Glu         #           270                                                               - Gly Tyr Tyr Pro Ile Asp Leu Asp Thr Arg Le - #u Gln Leu Gly Ala Pro         #       285                                                                   - Val Ser Arg Asn Phe Leu Thr Thr Gln His Va - #l Thr Val Ala Trp Asn         #   300                                                                       - Trp Val Pro Lys Ile Arg Glu Val Cys Thr Le - #u Ala Lys Trp Arg Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Asp Glu Ile Ile Arg Asp Glu Tyr Lys Gl - #y Ser Tyr Arg Phe Thr         #               335                                                           - Ala Lys Ser Ile Ser Ala Thr Phe Ile Ser As - #p Thr Thr Gln Phe Asp         #           350                                                               - Ile Asp Arg Val Lys Leu Ser Asp Cys Ala Ly - #s Arg Glu Ala Ile Glu         #       365                                                                   - Ala Ile Asp Lys Ile Tyr Lys Lys Lys Tyr As - #n Lys Thr His Ile Gln         #   380                                                                       - Thr Gly Glu Leu Glu Thr Tyr Leu Ala Arg Gl - #y Gly Phe Ile Ile Ala         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Phe Arg Pro Met Ile Ser Asn Glu Leu Ala Ly - #s Leu Tyr Ile Asn Glu         #               415                                                           - Leu Val Arg Ser Asn Arg Thr Val Asp Leu Ly - #s Ser Leu Leu Asn Pro         #           430                                                               - Ser Val Arg Gly Gly Ala Arg Lys Arg Arg Se - #r Val Glu Glu Asn Lys         #       445                                                                   - Arg Ser Lys Arg Asn Ile Glu Gly Gly Ile Gl - #u Asn Val Asn Asn Ser         #   460                                                                       - Thr Ile Ile Lys Thr Thr Ser Ser Val His Ph - #e Ala Met Leu Gln Phe         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ala Tyr Asp His Ile Gln Ser His Val Asn Gl - #u Met Leu Ser Arg Ile         #               495                                                           - Ala Thr Ala Trp Cys Asn Leu Gln Asn Lys Gl - #u Arg Thr Leu Trp Asn         #           510                                                               - Glu Val Met Lys Leu Asn Pro Thr Ser Val Al - #a Ser Val Ala Met Asp         #       525                                                                   - Gln Arg Val Ser Ala Arg Met Leu Gly Asp Va - #l Leu Ala Val Thr Gln         #   540                                                                       - Cys Val Asn Ile Ser Gly Ser Ser Val Phe Il - #e Gln Asn Ser Met Arg         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Val Leu Gly Ser Thr Thr Thr Cys Tyr Ser Ar - #g Pro Leu Ile Ser Phe         #               575                                                           - Lys Ala Leu Glu Asn Ser Thr Asn Tyr Ile Gl - #u Gly Gln Leu Gly Glu         #           590                                                               - Asn Asn Glu Leu Leu Val Glu Arg Lys Leu Il - #e Glu Pro Cys Thr Ala         #       605                                                                   - Asn His Lys Arg Tyr Phe Lys Phe Gly Ala As - #p Tyr Val Tyr Phe Glu         #   620                                                                       - Asn Tyr Ala Tyr Val Arg Lys Val Pro Leu As - #n Glu Ile Glu Met Ile         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ser Ala Tyr Val Asp Leu Asn Ile Thr Leu Le - #u Glu Asp Arg Glu Phe         #               655                                                           - Leu Pro Leu Glu Val Tyr Thr Arg Ala Glu Le - #u Glu Asp Thr Gly Leu         #           670                                                               - Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gl - #n Leu His Ala Leu Lys         #       685                                                                   - Phe Tyr Asp Ile Asp Ser Val Val Lys Val As - #p Asn Asn Val Val Ile         #   700                                                                       - Met Arg Gly Ile Ala Asn Phe Phe Gln Gly Le - #u Gly Asp Val Gly Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Phe Gly Lys Val Val Leu Gly Ala Ala As - #n Ala Val Ile Ala Thr         #               735                                                           - Val Ser Gly Val Ser Ser Phe Leu Asn Asn Pr - #o Phe Gly Ala Leu Ala         #           750                                                               - Val Gly Leu Leu Ile Leu Ala Gly Leu Phe Al - #a Ala Phe Leu Ala Tyr         #       765                                                                   - Arg Tyr Val Ser Lys Leu Lys Ser Asn Pro Me - #t Lys Ala Leu Tyr Pro         #   780                                                                       - Val Thr Thr Lys Asn Leu Lys Glu Ser Val Ly - #s Asn Gly Asn Ser Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asn Asn Ser Asp Gly Glu Glu Asn Asp Asp As - #n Ile Asp Glu Glu Lys         #               815                                                           - Leu Gln Gln Ala Lys Glu Met Ile Lys Tyr Me - #t Ser Leu Val Ser Ala         #           830                                                               - Met Glu Gln Gln Glu His Lys Ala Ile Lys Ly - #s Asn Ser Gly Pro Ala         #       845                                                                   - Leu Leu Ala Ser His Ile Thr Asn Leu Ser Le - #u Lys His Arg Gly Pro         #   860                                                                       - Lys Tyr Lys Arg Leu Lys Asn Val Asn Glu As - #n Glu Ser Lys Val             865                 8 - #70                 8 - #75                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1041 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ser Thr Arg Gly Asp Leu Gly Lys Arg Ar - #g Arg Gly Ser Arg Trp         #                15                                                           - Gln Gly His Ser Gly Tyr Phe Arg Gln Arg Cy - #s Phe Phe Pro Ser Leu         #            30                                                               - Leu Gly Ile Ala Ala Thr Gly Ser Arg His Gl - #y Asn Gly Ser Ser Gly         #        45                                                                   - Leu Thr Arg Leu Ala Arg Tyr Val Ser Phe Il - #e Trp Ile Val Leu Phe         #    60                                                                       - Leu Val Gly Pro Arg Pro Val Glu Gly Gln Se - #r Gly Ser Thr Ser Glu         #80                                                                           - Gln Pro Arg Arg Thr Val Ala Thr Pro Glu Va - #l Gly Gly Thr Pro Pro         #                95                                                           - Lys Pro Thr Thr Asp Pro Thr Asp Met Ser As - #p Met Arg Glu Ala Leu         #           110                                                               - Arg Ala Ser Gln Ile Glu Ala Asn Gly Pro Se - #r Thr Phe Tyr Met Cys         #       125                                                                   - Pro Pro Pro Ser Gly Ser Thr Val Val Arg Le - #u Glu Pro Pro Arg Ala         #   140                                                                       - Cys Pro Asp Tyr Lys Leu Gly Lys Asn Phe Th - #r Glu Gly Ile Ala Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Phe Lys Glu Asn Ile Ala Pro Tyr Lys Ph - #e Lys Ala Asn Ile Tyr         #               175                                                           - Tyr Lys Asn Ile Ile Met Thr Thr Val Trp Se - #r Gly Ser Ser Tyr Ala         #           190                                                               - Val Thr Thr Asn Arg Tyr Thr Asp Arg Val Pr - #o Val Lys Val Gln Glu         #       205                                                                   - Ile Thr Asp Leu Ile Asp Arg Arg Gly Met Cy - #s Leu Ser Lys Ala Asp         #   220                                                                       - Tyr Val Arg Asn Asn Tyr Gln Phe Thr Ala Ph - #e Asp Arg Asp Glu Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Arg Glu Leu Pro Leu Lys Pro Ser Lys Ph - #e Asn Thr Pro Gln Ser         #               255                                                           - Arg Gly Trp His Thr Tyr Lys Phe Lys Ala Th - #r Val Tyr Tyr Lys Asp         #           270                                                               - Val Ile Val Ser Thr Ala Trp Ala Gly Ser Se - #r Tyr Thr Gln Ile Thr         #       285                                                                   - Asn Arg Tyr Ala Asp Arg Val Pro Ile Pro Va - #l Ser Glu Ile Thr Asp         #   300                                                                       - Thr Ile Asp Lys Phe Gly Lys Cys Ser Ser Ly - #s Ala Thr Tyr Val Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Asn His Lys Val Glu Ala Phe Asn Glu As - #p Lys Asn Pro Gln Asp         #               335                                                           - Met Pro Leu Ile Ala Ser Lys Tyr Asn Ser Va - #l Gly Ser Lys Ala Trp         #           350                                                               - His Thr Thr Asn Glu Thr Tyr Thr Lys Ile Gl - #y Ala Ala Gly Phe His         #       365                                                                   - His Ser Gly Thr Ser Val Asn Cys Ile Val Gl - #u Glu Val Asp Ala Arg         #   380                                                                       - Ser Val Tyr Pro Tyr Asp Ser Phe Ala Ile Se - #r Thr Gly Asp Val Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - His Met Ser Pro Phe Phe Gly Leu Arg Asp Gl - #y Ala His Val Glu His         #               415                                                           - Thr Ser Tyr Ser Ser Asp Arg Phe Gln Gln Il - #e Glu Gly Tyr Tyr Pro         #           430                                                               - Ile Asp Leu Asp Thr Arg Leu Gln Leu Gly Al - #a Pro Val Ser Arg Asn         #       445                                                                   - Phe Leu Glu Thr Pro His Val Thr Val Ala Tr - #p Asn Trp Thr Pro Lys         #   460                                                                       - Cys Gly Arg Val Cys Thr Leu Ala Lys Trp Ar - #g Glu Ile Asp Glu Met         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Arg Asp Glu Tyr Gln Gly Ser Tyr Arg Ph - #e Thr Val Lys Thr Ile         #               495                                                           - Ser Ala Thr Phe Ile Ser Asn Thr Ser Gln Ph - #e Glu Ile Asn Arg Ile         #           510                                                               - Arg Leu Gly Asp Cys Ala Thr Lys Glu Ala Al - #a Glu Ala Ile Asp Arg         #       525                                                                   - Ile Tyr Lys Ser Lys Tyr Ser Lys Thr His Il - #e Gln Thr Gly Thr Leu         #   540                                                                       - Glu Thr Tyr Leu Ala Arg Gly Gly Phe Leu Il - #e Ala Phe Arg Pro Met         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Glu Leu Ala Lys Leu Tyr Ile As - #n Glu Leu Ala Arg Ser         #               575                                                           - Asn Arg Thr Val Asp Leu Ser Ala Leu Leu As - #n Pro Ser Gly Glu Thr         #           590                                                               - Val Gln Arg Thr Arg Gly Ser Val Pro Ser As - #n Gln His His Arg Ser         #       605                                                                   - Arg Arg Ser Thr Ile Glu Gly Gly Ile Glu Th - #r Val Asn Asn Ala Ser         #   620                                                                       - Leu Leu Lys Thr Thr Ser Ser Val Glu Phe Al - #a Met Ile Gln Phe Ala         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Tyr Asp Tyr Ile Gln Ala His Val Asn Glu Me - #t Leu Ser Arg Ile Ala         #               655                                                           - Thr Ala Trp Cys Thr Leu Gln Asn Arg Glu Hi - #s Val Leu Trp Thr Glu         #           670                                                               - Thr Leu Lys Leu Asn Pro Gly Gly Val Val Se - #r Met Ala Leu Glu Arg         #       685                                                                   - Arg Val Ser Ala Arg Leu Leu Gly Asp Ala Va - #l Ala Val Thr Gln Cys         #   700                                                                       - Val Asn Ile Ser Ser Gly His Val Tyr Ile Gl - #n Asn Ser Met Arg Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Gly Ser Ser Thr Thr Cys Tyr Ser Arg Pr - #o Leu Val Ser Phe Arg         #               735                                                           - Ala Leu Asn Asp Ser Glu Tyr Ile Glu Gly Gl - #n Leu Gly Glu Asn Asn         #           750                                                               - Asp Leu Leu Val Glu Arg Lys Leu Ile Glu Pr - #o Cys Thr Val Asn Asn         #       765                                                                   - Lys Arg Tyr Phe Lys Phe Gly Ala Asp Tyr Va - #l Tyr Phe Glu Asp Tyr         #   780                                                                       - Ala Tyr Val Arg Lys Val Pro Leu Ser Glu Il - #e Glu Leu Ile Ser Ala         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Tyr Val Asp Leu Asn Leu Thr Leu Leu Glu As - #p Arg Glu Phe Leu Pro         #               815                                                           - Leu Glu Val Tyr Thr Arg Ala Glu Leu Glu As - #p Thr Gly Leu Leu Asp         #           830                                                               - Tyr Ser Glu Ile Gln Arg Arg Asn Gln Leu Hi - #s Ala Leu Lys Phe Tyr         #       845                                                                   - Asp Ile Asp Ser Ile Val Arg Val Asp Asn As - #n Leu Val Ile Met Arg         #   860                                                                       - Gly Met Ala Asn Phe Phe Gln Gly Leu Gly As - #p Val Gly Ala Gly Phe         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Gly Lys Val Val Leu Gly Ala Ala Ser Ala Va - #l Ile Ser Thr Val Ser         #               895                                                           - Gly Val Ser Ser Phe Leu Asn Asn Pro Phe Gl - #y Ala Leu Ala Val Gly         #           910                                                               - Leu Leu Ile Leu Ala Gly Ile Val Ala Ala Ph - #e Leu Ala Tyr Arg Tyr         #       925                                                                   - Ile Ser Arg Leu Arg Ala Asn Pro Met Lys Al - #a Leu Tyr Pro Val Thr         #   940                                                                       - Thr Arg Asn Leu Lys Gln Thr Ala Lys Ser Pr - #o Ala Ser Thr Ala Gly         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Gly Asp Ser Asp Pro Gly Val Asp Asp Phe As - #p Glu Glu Lys Leu Met         #               975                                                           - Gln Ala Arg Glu Met Ile Lys Tyr Met Ser Le - #u Val Ser Ala Met Glu         #           990                                                               - Gln Gln Glu His Lys Ala Met Lys Lys Asn Ly - #s Gly Pro Ala Ile Leu         #      10050                                                                  - Thr Ser His Leu Thr Asn Met Ala Leu Arg Ar - #g Arg Gly Pro Lys Tyr         #  10205                                                                      - Gln Arg Leu Asn Asn Leu Asp Ser Gly Asp As - #p Thr Glu Thr Asn Leu         #               10401030 - #                1035                              - Val                                                                         - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 980 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Met Ser Ser Gly Cys Arg Ser Val Gly Gly Se - #r Thr Trp Gly Asn Trp         #                15                                                           - Arg Gly Asp Gly Gly Asp Leu Arg Gln Arg Ar - #g Val Leu Ser Pro Val         #            30                                                               - Cys Ser Ala Pro Ala Ala Gly Ser Trp Ile Gl - #y Ser Gln Leu Gly Asn         #        45                                                                   - Val Gly Asn Leu Leu Ala Thr Pro His Pro Le - #u Gly Lys Pro Ala Ser         #    60                                                                       - Ser Arg Val Gly Thr Ile Val Leu Ala Cys Le - #u Leu Leu Phe Gly Ser         #80                                                                           - Cys Val Val Arg Ala Val Pro Thr Thr Pro Se - #r Pro Pro Thr Ser Thr         #                95                                                           - Pro Thr Ser Met Ser Thr His Ser His Gly Th - #r Val Asp Pro Thr Leu         #           110                                                               - Leu Pro Thr Glu Thr Pro Asp Pro Leu Arg Le - #u Ala Val Arg Glu Ser         #       125                                                                   - Gly Ile Leu Ala Glu Asp Gly Asp Phe Tyr Th - #r Cys Pro Pro Pro Thr         #   140                                                                       - Gly Ser Thr Val Val Arg Ile Glu Pro Pro Ar - #g Thr Cys Pro Lys Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asp Leu Gly Arg Asn Phe Thr Glu Gly Ile Al - #a Val Ile Phe Lys Glu         #               175                                                           - Asn Ile Ala Pro Tyr Lys Phe Arg Ala Asn Va - #l Tyr Tyr Lys Asp Ile         #           190                                                               - Val Val Thr Arg Val Trp Lys Gly Tyr Ser Hi - #s Thr Ser Leu Ser Asp         #       205                                                                   - Arg Tyr Asn Asp Arg Val Pro Val Ser Val Gl - #u Glu Ile Phe Gly Leu         #   220                                                                       - Ile Asp Ser Lys Gly Lys Cys Ser Ser Lys Al - #a Glu Tyr Leu Arg Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Ile Met His His Ala Tyr His Asp Asp Gl - #u Asp Glu Val Glu Leu         #               255                                                           - Asp Leu Cys Arg Pro Ser Leu Gln Leu Arg Gl - #y Ala Arg Ala Trp Gln         #           270                                                               - Thr Thr Asn Asp Thr Thr Ser Tyr Val Gly Tr - #p Met Pro Trp Arg His         #       285                                                                   - Tyr Thr Ser Thr Ser Val Asn Cys Ile Val Gl - #u Glu Val Glu Ala Arg         #   300                                                                       - Ser Val Tyr Pro Tyr Asp Ser Phe Ala Leu Se - #r Thr Gly Asp Ile Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Ala Ser Pro Phe Tyr Gly Leu Arg Ala Al - #a Ala Arg Ile Glu His         #               335                                                           - Asn Ser Tyr Ala Gln Glu Arg Phe Arg Gln Va - #l Glu Gly Tyr Arg Pro         #           350                                                               - Arg Asp Leu Asp Ser Lys Leu Gln Ala Glu Gl - #u Pro Val Thr Lys Asn         #       365                                                                   - Phe Ile Thr Thr Pro His Val Thr Val Ser Tr - #p Asn Trp Thr Glu Lys         #   380                                                                       - Lys Val Glu Ala Cys Thr Leu Thr Lys Trp Ly - #s Glu Val Asp Glu Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Arg Asp Glu Phe Arg Gly Ser Tyr Arg Ph - #e Thr Ile Arg Ser Ile         #               415                                                           - Ser Ser Thr Phe Ile Ser Asn Thr Thr Gln Ph - #e Lys Leu Glu Ser Ala         #           430                                                               - Pro Leu Thr Glu Cys Val Ser Lys Glu Ala Ly - #s Glu Ala Ile Asp Ser         #       445                                                                   - Ile Tyr Lys Lys Gln Tyr Glu Ser Thr His Va - #l Phe Ser Gly Asp Val         #   460                                                                       - Glu Tyr Tyr Leu Ala Arg Gly Gly Phe Leu Il - #e Ala Phe Arg Pro Met         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Ser Asn Glu Leu Ala Arg Leu Tyr Leu As - #n Glu Leu Val Arg Ser         #               495                                                           - Asn Arg Thr Tyr Asp Leu Lys Asn Leu Leu As - #n Pro Asn Ala Asn Asn         #           510                                                               - Asn Asn Asn Thr Thr Arg Arg Arg Arg Ser Le - #u Leu Ser Val Pro Glu         #       525                                                                   - Pro Gln Pro Thr Gln Asp Gly Val His Arg Gl - #u Gln Ile Leu His Arg         #   540                                                                       - Leu His Lys Arg Ala Val Glu Ala Thr Ala Gl - #y Thr Asp Ser Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Val Thr Ala Lys Gln Leu Glu Leu Ile Lys Th - #r Thr Ser Ser Ile Glu         #               575                                                           - Phe Ala Met Leu Gln Phe Ala Tyr Asp His Il - #e Gln Ser His Val Asn         #           590                                                               - Glu Met Leu Ser Arg Ile Ala Thr Ala Trp Cy - #s Thr Leu Gln Asn Lys         #       605                                                                   - Glu Arg Thr Leu Trp Asn Glu Met Val Lys Il - #e Asn Pro Ser Ala Ile         #   620                                                                       - Val Ser Ala Thr Leu Asp Glu Arg Val Ala Al - #a Arg Val Leu Gly Asp         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Val Ile Ala Ile Thr His Cys Ala Lys Ile Gl - #u Gly Asn Val Tyr Leu         #               655                                                           - Gln Asn Ser Met Arg Ser Met Asp Ser Asn Th - #r Cys Tyr Ser Arg Pro         #           670                                                               - Pro Val Thr Phe Thr Ile Thr Lys Asn Ala As - #n Asn Arg Gly Ser Ile         #       685                                                                   - Glu Gly Gln Leu Gly Glu Glu Asn Glu Ile Ph - #e Thr Glu Arg Lys Leu         #   700                                                                       - Ile Glu Pro Cys Ala Leu Asn Gln Lys Arg Ty - #r Phe Lys Phe Gly Lys         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Glu Tyr Val Tyr Tyr Glu Asn Tyr Thr Phe Va - #l Arg Lys Val Pro Pro         #               735                                                           - Thr Glu Ile Glu Val Ile Ser Thr Tyr Val Gl - #u Leu Asn Leu Thr Leu         #           750                                                               - Leu Glu Asp Arg Glu Phe Leu Pro Leu Glu Va - #l Tyr Thr Arg Ala Glu         #       765                                                                   - Leu Glu Asp Thr Gly Leu Leu Asp Tyr Ser Gl - #u Ile Gln Arg Arg Asn         #   780                                                                       - Gln Leu His Ala Leu Arg Phe Tyr Asp Ile As - #p Ser Val Val Asn Val         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asp Asn Thr Ala Val Ile Met Gln Gly Ile Al - #a Ser Phe Phe Lys Gly         #               815                                                           - Leu Gly Lys Val Gly Glu Ala Val Gly Thr Le - #u Val Leu Gly Ala Ala         #           830                                                               - Gly Ala Val Val Ser Thr Val Ser Gly Ile Al - #a Ser Phe Leu Asn Asn         #       845                                                                   - Pro Phe Gly Gly Leu Ala Ile Gly Leu Leu Va - #l Ile Ala Gly Leu Val         #   860                                                                       - Ala Ala Phe Phe Ala Tyr Arg Tyr Val Met Gl - #n Ile Arg Ser Asn Pro         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Met Lys Ala Leu Tyr Pro Ile Thr Thr Lys Al - #a Leu Lys Asn Lys Ala         #               895                                                           - Lys Thr Ser Tyr Gly Gln Asn Glu Glu Asp As - #p Gly Ser Asp Phe Asp         #           910                                                               - Glu Ala Lys Leu Glu Glu Ala Arg Glu Met Il - #e Lys Tyr Met Ser Met         #       925                                                                   - Val Ser Ala Leu Glu Lys Gln Glu Lys Lys Al - #a Ile Lys Lys Asn Ser         #   940                                                                       - Gly Val Gly Leu Ile Ala Ser Asn Val Ser Ly - #s Leu Ala Leu Arg Arg         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Arg Gly Pro Lys Tyr Thr Arg Leu Gln Gln As - #n Asp Thr Met Glu Asn         #               975                                                           - Glu Lys Met Val                                                                         980                                                               - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 913 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Pro Ala Gly Gly Gly Leu Trp Arg Gly Pr - #o Arg Gly His Arg Pro         #                15                                                           - Gly His His Gly Gly Ala Gly Leu Gly Arg Le - #u Trp Pro Ala Pro His         #            30                                                               - His Ala Ala Ala Ala Arg Gly Ala Val Ala Le - #u Ala Leu Leu Leu Leu         #        45                                                                   - Ala Leu Ala Ala Ala Pro Pro Cys Gly Ala Al - #a Ala Val Thr Arg Ala         #    60                                                                       - Ala Ser Ala Ser Pro Thr Pro Gly Thr Gly Al - #a Thr Pro Asn Asp Val         #80                                                                           - Ser Ala Glu Ala Ser Leu Glu Glu Ile Glu Al - #a Phe Ser Pro Gly Pro         #                95                                                           - Ser Glu Ala Pro Asp Gly Glu Tyr Gly Asp Le - #u Asp Ala Arg Thr Ala         #           110                                                               - Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Ph - #e Tyr Val Cys Pro Pro         #       125                                                                   - Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pr - #o Glu Gln Ala Cys Pro         #   140                                                                       - Glu Tyr Ser Gln Gly Arg Asn Phe Thr Glu Gl - #y Ile Ala Val Leu Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Glu Asn Ile Ala Pro His Lys Phe Lys Al - #a His Ile Tyr Tyr Lys         #               175                                                           - Asn Val Ile Val Thr Thr Val Trp Ser Gly Se - #r Thr Tyr Ala Ala Ile         #           190                                                               - Thr Asn Arg Phe Thr Asp Arg Val Pro Val Pr - #o Val Gln Glu Ile Thr         #       205                                                                   - Asp Val Ile Asp Arg Arg Gly Lys Cys Val Se - #r Lys Ala Glu Tyr Val         #   220                                                                       - Arg Asn Asn His Lys Val Thr Ala Phe Asp Ar - #g Asp Glu Asn Pro Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Glu Val Asp Leu Arg Pro Ser Arg Leu Asn Al - #a Leu Gly Thr Arg Gly         #               255                                                           - Trp His Thr Thr Asn Asp Thr Tyr Thr Lys Il - #e Gly Ala Ala Gly Phe         #           270                                                               - Tyr His Thr Gly Thr Ser Val Asn Cys Ile Va - #l Glu Glu Val Glu Ala         #       285                                                                   - Arg Ser Val Tyr Pro Tyr Asp Ser Phe Ala Le - #u Ser Thr Gly Asp Ile         #   300                                                                       - Val Tyr Met Ser Pro Phe Tyr Gly Leu Arg Gl - #u Gly Ala His Gly Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - His Ile Gly Tyr Ala Pro Gly Arg Phe Gln Gl - #n Val Glu His Tyr Tyr         #               335                                                           - Pro Ile Asp Leu Asp Ser Arg Leu Arg Ala Se - #r Glu Ser Val Thr Arg         #           350                                                               - Asn Phe Leu Arg Thr Pro His Phe Thr Val Al - #a Trp Asp Trp Ala Pro         #       365                                                                   - Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Tr - #p Arg Glu Ala Glu Glu         #   380                                                                       - Met Thr Arg Asp Glu Thr Arg Asp Gly Ser Ph - #e Arg Phe Thr Ser Arg         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ala Leu Gly Ala Ser Phe Val Ser Asp Val Th - #r Gln Leu Asp Leu Gln         #               415                                                           - Arg Val His Leu Gly Asp Cys Val Leu Arg Gl - #u Ala Ser Glu Ala Ile         #           430                                                               - Asp Ala Ile Tyr Arg Arg Arg Tyr Asn Ser Th - #r His Val Leu Ala Gly         #       445                                                                   - Asp Arg Pro Glu Val Tyr Leu Ala Arg Gly Gl - #y Phe Val Val Ala Phe         #   460                                                                       - Arg Pro Leu Ile Ser Asn Glu Leu Ala Gln Le - #u Tyr Ala Arg Glu Leu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Glu Arg Leu Gly Leu Ala Gly Val Val Gly Pr - #o Ala Ala Pro Ala Ala         #               495                                                           - Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Al - #a Gly Thr Pro Glu Pro         #           510                                                               - Pro Ala Val Asn Gly Thr Gly His Leu Arg Il - #e Thr Thr Gly Ser Ala         #       525                                                                   - Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asp Hi - #s Ile Gln Ala His Val         #   540                                                                       - Asn Asp Met Leu Gly Arg Ile Ala Ala Ala Tr - #p Cys Glu Leu Gln Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Lys Asp Arg Thr Leu Trp Ser Glu Met Ser Ar - #g Leu Asn Pro Ser Ala         #               575                                                           - Val Ala Thr Ala Ala Leu Gly Gln Arg Val Se - #r Ala Arg Met Leu Gly         #           590                                                               - Asp Val Met Ala Ile Ser Arg Cys Val Glu Va - #l Arg Gly Gly Val Tyr         #       605                                                                   - Val Gln Asn Ser Met Arg Val Pro Gly Glu Ar - #g Gly Thr Cys Tyr Ser         #   620                                                                       - Arg Pro Leu Val Thr Phe Glu His Asn Gly Th - #r Gly Val Ile Glu Gly         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Gln Leu Gly Asp Asp Asn Glu Leu Leu Ile Se - #r Arg Asp Leu Ile Glu         #               655                                                           - Pro Cys Thr Gly Asn His Arg Arg Tyr Phe Ly - #s Leu Gly Ser Gly Tyr         #           670                                                               - Val Tyr Tyr Glu Asp Tyr Asn Tyr Val Arg Me - #t Val Glu Val Pro Glu         #       685                                                                   - Thr Ile Ser Thr Arg Val Thr Leu Asn Leu Th - #r Leu Leu Glu Asp Arg         #   700                                                                       - Glu Phe Leu Pro Leu Glu Val Tyr Thr Arg Gl - #u Glu Leu Ala Asp Thr         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Leu Leu Asp Tyr Ser Glu Ile Gln Arg Ar - #g Asn Gln Leu His Ala         #               735                                                           - Leu Lys Phe Tyr Asp Ile Asp Arg Val Val Ly - #s Val Asp His Asn Val         #           750                                                               - Val Leu Leu Arg Gly Ile Ala Asn Phe Phe Gl - #n Gly Leu Gly Asp Val         #       765                                                                   - Gly Ala Ala Val Gly Lys Val Val Leu Gly Al - #a Thr Gly Ala Val Ile         #   780                                                                       - Ser Ala Val Gly Gly Met Val Ser Phe Leu Se - #r Asn Pro Phe Gly Ala         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Leu Ala Ile Gly Leu Leu Val Leu Ala Gly Le - #u Val Ala Ala Phe Leu         #               815                                                           - Ala Tyr Arg His Ile Ser Arg Leu Arg Arg As - #n Pro Met Lys Ala Leu         #           830                                                               - Tyr Pro Val Thr Thr Lys Thr Leu Lys Glu As - #p Gly Val Asp Glu Gly         #       845                                                                   - Asp Val Asp Glu Ala Lys Leu Asp Gln Ala Ar - #g Asp Met Ile Arg Tyr         #   860                                                                       - Met Ser Ile Val Ser Ala Leu Glu Gln Gln Gl - #u His Lys Ala Arg Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Ar - #g Val Gly Ala Met Ala         #               895                                                           - Thr Arg Arg Arg His Tyr Gln Arg Leu Glu Se - #r Glu Asp Pro Asp Ala         #           910                                                               - Leu                                                                         - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 868 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Met Phe Val Thr Ala Val Val Ser Val Ser Pr - #o Ser Ser Phe Tyr Glu         #                15                                                           - Ser Leu Gln Val Glu Pro Thr Gln Ser Glu As - #p Ile Thr Arg Ser Ala         #            30                                                               - His Leu Gly Asp Gly Asp Glu Ile Arg Glu Al - #a Ile His Lys Ser Gln         #        45                                                                   - Asp Ala Glu Thr Lys Pro Thr Phe Tyr Val Cy - #s Pro Pro Pro Thr Gly         #    60                                                                       - Ser Thr Ile Val Arg Leu Glu Pro Thr Arg Th - #r Cys Pro Asp Tyr His         #80                                                                           - Leu Gly Lys Asn Phe Thr Glu Gly Ile Ala Va - #l Val Tyr Lys Glu Asn         #                95                                                           - Ile Ala Ala Tyr Lys Phe Lys Ala Thr Val Ty - #r Tyr Lys Asp Val Ile         #           110                                                               - Val Ser Thr Ala Trp Ala Gly Ser Ser Tyr Th - #r Gln Ile Thr Asn Arg         #       125                                                                   - Tyr Ala Asp Arg Val Pro Ile Pro Val Ser Gl - #u Ile Thr Asp Thr Ile         #   140                                                                       - Asp Lys Phe Gly Lys Cys Ser Ser Lys Ala Th - #r Tyr Val Arg Asn Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - His Lys Val Glu Ala Phe Asn Glu Asp Lys As - #n Pro Gln Asp Met Pro         #               175                                                           - Leu Ile Ala Ser Lys Tyr Asn Ser Val Gly Se - #r Lys Ala Trp His Thr         #           190                                                               - Thr Asn Asp Thr Tyr Met Val Ala Gly Thr Pr - #o Gly Thr Tyr Arg Thr         #       205                                                                   - Gly Thr Ser Val Asn Cys Ile Ile Glu Glu Va - #l Glu Ala Arg Ser Ile         #   220                                                                       - Phe Pro Tyr Asp Ser Phe Gly Leu Ser Thr Gl - #y Asp Ile Ile Tyr Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Pro Phe Phe Gly Leu Arg Asp Gly Ala Ty - #r Arg Glu His Ser Asn         #               255                                                           - Tyr Ala Met Asp Arg Phe His Gln Phe Glu Gl - #y Tyr Arg Gln Arg Asp         #           270                                                               - Leu Asp Thr Arg Ala Leu Leu Glu Pro Ala Al - #a Arg Asn Phe Leu Val         #       285                                                                   - Thr Pro His Leu Thr Val Gly Trp Asn Trp Ly - #s Pro Lys Arg Thr Glu         #   300                                                                       - Val Cys Ser Leu Val Lys Trp Arg Glu Val Gl - #u Asp Val Val Arg Asp         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Tyr Ala His Asn Phe Arg Phe Thr Met Ly - #s Thr Leu Ser Thr Thr         #               335                                                           - Phe Ile Ser Glu Thr Asn Glu Phe Asn Leu As - #n Gln Ile His Leu Ser         #           350                                                               - Gln Cys Val Lys Glu Glu Ala Arg Ala Ile Il - #e Asn Arg Ile Tyr Thr         #       365                                                                   - Thr Arg Tyr Asn Ser Ser His Val Arg Thr Gl - #y Asp Ile Gln Thr Tyr         #   380                                                                       - Leu Ala Arg Gly Gly Phe Val Val Val Phe Gl - #n Pro Leu Leu Ser Asn         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Leu Ala Arg Leu Tyr Leu Gln Glu Leu Va - #l Arg Glu Asn Thr Asn         #               415                                                           - His Ser Pro Gln Lys His Pro Thr Arg Asn Th - #r Arg Ser Arg Arg Ser         #           430                                                               - Val Pro Val Glu Leu Arg Ala Asn Arg Thr Il - #e Thr Thr Thr Ser Ser         #       445                                                                   - Val Glu Phe Ala Met Leu Gln Phe Thr Tyr As - #p His Ile Gln Glu His         #   460                                                                       - Val Asn Glu Met Leu Ala Arg Ile Ser Ser Se - #r Trp Cys Gln Leu Gln         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Arg Glu Arg Ala Leu Trp Ser Gly Leu Ph - #e Pro Ile Asn Pro Ser         #               495                                                           - Ala Leu Ala Ser Thr Ile Leu Asp Gln Arg Va - #l Lys Ala Arg Ile Leu         #           510                                                               - Gly Asp Val Ile Ser Val Ser Asn Cys Pro Gl - #u Leu Gly Ser Asp Thr         #       525                                                                   - Arg Ile Ile Leu Gln Asn Ser Met Arg Val Se - #r Gly Ser Thr Thr Arg         #   540                                                                       - Cys Tyr Ser Arg Pro Leu Ile Ser Ile Val Se - #r Leu Asn Gly Ser Gly         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Thr Val Glu Gly Gln Leu Gly Thr Asp Asn Gl - #u Leu Ile Met Ser Arg         #               575                                                           - Asp Leu Leu Glu Pro Cys Val Ala Asn His Ly - #s Arg Tyr Phe Leu Phe         #           590                                                               - Gly His His Tyr Val Tyr Tyr Glu Asp Tyr Ar - #g Tyr Val Arg Glu Ile         #       605                                                                   - Ala Val His Asp Val Gly Met Ile Ser Thr Ty - #r Val Asp Leu Asn Leu         #   620                                                                       - Thr Leu Leu Lys Asp Arg Glu Phe Met Pro Le - #u Gln Val Tyr Thr Arg         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Asp Glu Leu Arg Asp Thr Gly Leu Leu Asp Ty - #r Ser Glu Ile Gln Arg         #               655                                                           - Arg Asn Gln Met His Ser Leu Arg Phe Tyr As - #p Ile Asp Lys Val Val         #           670                                                               - Gln Tyr Asp Ser Gly Thr Ala Ile Met Gln Gl - #y Met Ala Gln Phe Phe         #       685                                                                   - Gln Gly Leu Gly Thr Ala Gly Gln Ala Val Gl - #y His Val Val Leu Gly         #   700                                                                       - Ala Thr Gly Ala Leu Leu Ser Thr Val His Gl - #y Phe Thr Thr Phe Leu         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Le - #u Leu Val Leu Ala Gly         #               735                                                           - Leu Val Ala Ala Phe Phe Ala Tyr Arg Tyr Va - #l Leu Lys Leu Lys Thr         #           750                                                               - Ser Pro Met Lys Ala Leu Tyr Pro Leu Thr Th - #r Lys Gly Leu Lys Gln         #       765                                                                   - Leu Pro Glu Gly Met Asp Pro Phe Ala Glu Ly - #s Pro Asn Ala Thr Asp         #   780                                                                       - Thr Pro Ile Glu Glu Ile Gly Asp Ser Gln As - #n Thr Glu Pro Ser Val         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asn Ser Gly Phe Asp Pro Asp Lys Phe Arg Gl - #u Ala Gln Glu Met Ile         #               815                                                           - Lys Tyr Met Thr Leu Val Ser Ala Ala Glu Ar - #g Gln Glu Ser Lys Ala         #           830                                                               - Arg Lys Lys Asn Lys Thr Ser Ala Leu Leu Th - #r Ser Arg Leu Thr Gly         #       845                                                                   - Leu Ala Leu Arg Asn Arg Arg Gly Tyr Ser Ar - #g Val Arg Thr Glu Asn         #   860                                                                       - Val Thr Gly Val                                                             865                                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 903 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met His Gln Gly Ala Pro Ser Trp Gly Arg Ar - #g Trp Phe Val Val Trp         #                15                                                           - Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Va - #l Ala Ser Ala Ala Pro         #            30                                                               - Ser Ser Pro Gly Thr Pro Gly Val Ala Arg As - #p Pro Gly Gly Glu Arg         #        45                                                                   - Gly Pro Cys His Ser Gly Ala Ala Ala Leu Gl - #y Ala Ala Pro Thr Gly         #    60                                                                       - Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Ly - #s Asn Pro Thr Pro Pro         #80                                                                           - Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Al - #a Gly His Ala Thr Leu         #                95                                                           - Arg Glu His Leu Arg Asp Ile Lys Ala Glu As - #n Thr Asp Ala Asn Phe         #           110                                                               - Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Va - #l Val Gln Phe Glu Gln         #       125                                                                   - Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gl - #n Asn Tyr Thr Glu Gly         #   140                                                                       - Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pr - #o Tyr Lys Phe Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gl - #n Val Trp Phe Gly His         #               175                                                           - Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu As - #p Arg Ala Pro Val Pro         #           190                                                               - Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Ly - #s Gly Val Cys Arg Ser         #       205                                                                   - Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Th - #r Thr Ala Phe His Arg         #   220                                                                       - Asp Asp His Glu Thr Asp Met Glu Leu Lys Pr - #o Ala Asn Ala Ala Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Thr Ser Arg Gly Trp His Thr Thr Asp Le - #u Lys Tyr Asn Pro Ser         #               255                                                           - Arg Val Glu Ala Phe His Arg Tyr Gly Thr Th - #r Val Asn Cys Ile Val         #           270                                                               - Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Ty - #r Asp Glu Phe Val Leu         #       285                                                                   - Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Ph - #e Tyr Gly Tyr Arg Glu         #   300                                                                       - Gly Ser His Thr Glu His Thr Thr Tyr Ala Al - #a Asp Arg Phe Lys Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Th - #r Lys Ala Arg Ala Thr         #               335                                                           - Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pr - #o Lys Phe Thr Val Ala         #           350                                                               - Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cy - #s Thr Met Thr Lys Trp         #       365                                                                   - Gln Glu Val Asp Glu Met Leu Arg Ser Glu Ty - #r Gly Gly Ser Phe Arg         #   380                                                                       - Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Th - #r Thr Asn Leu Thr Glu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cy - #s Ile Gly Lys Asp Ala         #               415                                                           - Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Ar - #g Tyr Asn Ala Thr His         #           430                                                               - Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Al - #a Asn Gly Gly Phe Leu         #       445                                                                   - Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Le - #u Ala Glu Leu Tyr Val         #   460                                                                       - Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pr - #o Pro Asn Pro Thr Pro         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Va - #l Glu Arg Ile Lys Thr         #               495                                                           - Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Ph - #e Thr Tyr Asn His Ile         #           510                                                               - Gln Arg His Val Asn Asp Met Leu Gly Arg Va - #l Ala Ile Ala Trp Cys         #       525                                                                   - Glu Leu Gln Asn His Glu Leu Thr Leu Trp As - #n Glu Ala Arg Lys Leu         #   540                                                                       - Asn Pro Asn Ala Ile Ala Ser Val Thr Val Gl - #y Arg Arg Val Ser Ala         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Arg Met Leu Gly Asp Val Met Ala Val Ser Th - #r Cys Val Pro Val Ala         #               575                                                           - Ala Asp Asn Val Ile Val Gln Asn Ser Met Ar - #g Ile Ser Ser Arg Pro         #           590                                                               - Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Ph - #e Arg Tyr Glu Asp Gln         #       605                                                                   - Gly Pro Leu Val Glu Gly Gln Leu Gly Glu As - #n Asn Glu Leu Arg Leu         #   620                                                                       - Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gl - #y His Arg Arg Tyr Phe         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Gl - #u Tyr Ala Tyr Ser His         #               655                                                           - Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Se - #r Thr Phe Ile Asp Leu         #           670                                                               - Asn Ile Thr Met Leu Glu Asp His Glu Phe Va - #l Pro Leu Glu Val Tyr         #       685                                                                   - Thr Arg His Glu Ile Lys Asp Ser Gly Leu Le - #u Asp Tyr Thr Glu Val         #   700                                                                       - Gln Arg Arg Asn Gln Leu His Asp Leu Arg Ph - #e Ala Asp Ile Asp Thr         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Val Ile His Ala Asp Ala Asn Ala Ala Met Ph - #e Ala Gly Leu Gly Ala         #               735                                                           - Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Al - #a Val Gly Lys Val Val         #           750                                                               - Met Gly Leu Val Gly Gly Val Val Ser Ala Va - #l Ser Gly Val Ser Ser         #       765                                                                   - Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Va - #l Gly Leu Leu Val Leu         #   780                                                                       - Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Ar - #g Tyr Val Met Arg Leu         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Le - #u Thr Thr Lys Glu Leu         #               815                                                           - Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Gl - #u Gly Glu Glu Gly Gly         #           830                                                               - Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Ar - #g Glu Met Ile Arg Tyr         #       845                                                                   - Met Ala Leu Val Ser Ala Met Glu Arg Thr Gl - #u His Lys Ala Lys Lys         #   860                                                                       - Lys Gly Thr Ser Arg Leu Leu Ser Ala Lys Va - #l Thr Asp Met Val Met         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Va - #l Pro Asn Lys Asp Gly         #               895                                                           - Asp Ala Asp Glu Asp Asp Leu                                                             900                                                               - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 906 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Met Glu Ser Arg Ile Trp Cys Leu Val Val Cy - #s Val Asn Leu Cys Ile         #                15                                                           - Val Cys Leu Gly Ala Ala Val Ser Ser Ser Se - #r Thr Ser His Ala Thr         #            30                                                               - Ser Ser Thr His Asn Gly Ser His Thr Ser Ar - #g Thr Thr Ser Ala Gln         #        45                                                                   - Thr Arg Ser Val Tyr Ser Gln His Val Thr Se - #r Ser Glu Ala Val Ser         #    60                                                                       - His Arg Ala Asn Glu Thr Ile Tyr Asn Thr Th - #r Leu Lys Tyr Gly Asp         #80                                                                           - Val Val Gly Val Asn Thr Thr Lys Tyr Pro Ty - #r Arg Val Cys Ser Met         #                95                                                           - Ala Gln Gly Thr Asp Leu Ile Arg Phe Glu Ar - #g Asn Ile Ile Cys Thr         #           110                                                               - Ser Met Lys Pro Ile Asn Glu Asp Leu Asp Gl - #u Gly Ile Met Val Val         #       125                                                                   - Tyr Lys Arg Asn Ile Val Ala His Thr Phe Ly - #s Val Arg Val Tyr Gln         #   140                                                                       - Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Ty - #r Ile Tyr Thr Thr Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Gly Ser Asn Thr Glu Tyr Val Ala Pr - #o Pro Met Trp Glu Ile         #               175                                                           - His His Ile Asn Lys Phe Ala Gln Cys Tyr Se - #r Ser Tyr Ser Arg Val         #           190                                                               - Ile Gly Gly Thr Val Phe Val Ala Tyr His Ar - #g Asp Ser Tyr Glu Asn         #       205                                                                   - Lys Thr Met Gln Leu Ile Pro Asp Asp Tyr Se - #r Asn Thr His Ser Thr         #   220                                                                       - Arg Tyr Val Thr Val Lys Asp Gln Trp His Se - #r Arg Gly Ser Thr Trp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Me - #t Leu Thr Ile Thr Thr         #               255                                                           - Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Al - #a Thr Ser Thr Gly Asp         #           270                                                               - Val Val Tyr Ile Ser Pro Phe Tyr Asn Gly Th - #r Asn Arg Asn Ala Ser         #       285                                                                   - Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe Il - #e Phe Pro Asn Tyr Thr         #   300                                                                       - Ile Val Ser Asp Phe Gly Arg Pro Asn Ala Al - #a Pro Glu Thr His Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Va - #l Ile Ser Trp Asp Ile         #               335                                                           - Gln Asp Glu Lys Asn Val Thr Cys Gln Leu Th - #r Phe Trp Glu Ala Ser         #           350                                                               - Glu Arg Thr Ile Arg Ser Glu Ala Glu Asp Se - #r Tyr His Phe Ser Ser         #       365                                                                   - Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Ly - #s Gln Glu Val Asn Met         #   380                                                                       - Ser Asp Ser Ala Leu Asp Cys Val Arg Asp Gl - #u Ala Ile Asn Lys Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gln Gln Ile Phe Asn Thr Ser Tyr Asn Gln Th - #r Tyr Glu Lys Tyr Gly         #               415                                                           - Asn Val Ser Val Phe Glu Thr Ser Gly Gly Le - #u Val Val Phe Trp Gln         #           430                                                               - Gly Ile Lys Gln Lys Ser Leu Val Glu Leu Gl - #u Arg Leu Ala Asn Arg         #       445                                                                   - Ser Ser Leu Asn Ile Thr His Arg Thr Arg Ar - #g Ser Thr Ser Asp Asn         #   460                                                                       - Asn Thr Thr His Leu Ser Ser Met Glu Ser Va - #l His Asn Leu Val Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ala Gln Leu Gln Phe Thr Tyr Asp Thr Leu Ar - #g Gly Tyr Ile Asn Arg         #               495                                                           - Ala Leu Ala Gln Ile Ala Glu Ala Trp Cys Va - #l Asp Gln Arg Arg Thr         #           510                                                               - Leu Glu Val Phe Lys Glu Leu Ser Lys Ile As - #n Pro Ser Ala Ile Leu         #       525                                                                   - Ser Ala Ile Tyr Asn Lys Pro Ile Ala Ala Ar - #g Phe Met Gly Asp Val         #   540                                                                       - Leu Gly Leu Ala Ser Cys Val Thr Ile Asn Gl - #n Thr Ser Val Lys Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gl - #y Arg Cys Tyr Ser Arg         #               575                                                           - Pro Val Val Ile Phe Asn Phe Ala Asn Ser Se - #r Tyr Val Gln Tyr Gly         #           590                                                               - Gln Leu Gly Glu Asp Asn Glu Ile Leu Leu Gl - #y Asn His Arg Thr Glu         #       605                                                                   - Glu Cys Gln Leu Pro Ser Leu Lys Ile Phe Il - #e Ala Gly Asn Ser Ala         #   620                                                                       - Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Me - #t Ile Asp Leu Ser Ser         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ile Ser Thr Val Asp Ser Met Ile Ala Leu As - #p Ile Asp Pro Leu Glu         #               655                                                           - Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr Se - #r Gln Lys Glu Leu Arg         #           670                                                               - Ser Ser Asn Val Phe Asp Leu Glu Glu Ile Me - #t Arg Glu Phe Asn Ser         #       685                                                                   - Tyr Lys Gln Arg Val Lys Tyr Val Glu Asp Ly - #s Val Val Asp Pro Leu         #   700                                                                       - Pro Pro Tyr Leu Lys Gly Leu Asp Asp Leu Me - #t Ser Gly Leu Gly Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Ala Gly Lys Ala Val Gly Val Ala Ile Gly Al - #a Val Gly Gly Ala Val         #               735                                                           - Ala Ser Val Val Glu Gly Val Ala Thr Phe Le - #u Lys Asn Pro Phe Gly         #           750                                                               - Ala Phe Thr Ile Ile Leu Val Ala Ile Ala Va - #l Val Ile Ile Thr Tyr         #       765                                                                   - Leu Ile Tyr Thr Arg Gln Arg Arg Leu Cys Th - #r Gln Pro Leu Gln Asn         #   780                                                                       - Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Th - #r Thr Val Thr Ser Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Ser Thr Lys Asp Thr Ser Leu Gln Ala Pro Pr - #o Ser Tyr Glu Glu Ser         #               815                                                           - Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pr - #o Pro Ser Ser Asp Ala         #           830                                                               - Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gl - #n Ala Tyr Gln Met Leu         #       845                                                                   - Leu Ala Leu Ala Arg Leu Asp Ala Glu Gln Ar - #g Ala Gln Gln Asn Gly         #   860                                                                       - Thr Asp Ser Leu Asp Gly Gln Thr Gly Thr Gl - #n Asp Lys Gly Gln Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Pro Asn Leu Leu Asp Arg Leu Arg His Arg Ly - #s Asn Gly Tyr Arg His         #               895                                                           - Leu Lys Asp Ser Asp Glu Glu Glu Asn Val                                     #           905                                                               - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 857 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Thr Arg Arg Arg Val Leu Ser Val Val Va - #l Leu Leu Ala Ala Leu         #                15                                                           - Ala Cys Arg Leu Gly Ala Gln Thr Pro Glu Gl - #n Pro Ala Pro Pro Ala         #            30                                                               - Thr Thr Val Gln Pro Thr Ala Thr Arg Gln Gl - #n Thr Ser Phe Pro Phe         #        45                                                                   - Arg Val Cys Glu Leu Ser Ser His Gly Asp Le - #u Phe Arg Phe Ser Ser         #    60                                                                       - Asp Ile Gln Cys Pro Ser Phe Gly Thr Arg Gl - #u Asn His Thr Glu Gly         #80                                                                           - Leu Leu Met Val Phe Lys Asp Asn Ile Ile Pr - #o Tyr Ser Phe Lys Val         #                95                                                           - Arg Ser Tyr Thr Lys Ile Val Thr Asn Ile Le - #u Ile Tyr Asn Gly Trp         #           110                                                               - Tyr Ala Asp Ser Val Thr Asn Arg His Glu Gl - #u Lys Phe Ser Val Asp         #       125                                                                   - Ser Tyr Glu Thr Asp Gln Met Asp Thr Ile Ty - #r Gln Cys Tyr Asn Ala         #   140                                                                       - Val Lys Met Thr Lys Asp Gly Leu Thr Arg Va - #l Tyr Val Asp Arg Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Val Asn Ile Thr Val Asn Leu Lys Pro Th - #r Gly Gly Leu Ala Asn         #               175                                                           - Gly Val Arg Arg Tyr Ala Ser Gln Thr Glu Le - #u Tyr Asp Ala Pro Gly         #           190                                                               - Trp Leu Ile Trp Thr Tyr Arg Thr Arg Thr Th - #r Val Asn Cys Leu Ile         #       205                                                                   - Thr Asp Met Met Ala Lys Ser Asn Ser Pro Ph - #e Asp Phe Phe Val Thr         #   220                                                                       - Thr Thr Gly Gln Thr Val Glu Met Ser Pro Ph - #e Tyr Asp Gly Lys Asn         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Lys Glu Thr Phe His Glu Arg Ala Asp Ser Ph - #e His Val Arg Thr Asn         #               255                                                           - Tyr Lys Ile Val Asp Tyr Asp Asn Arg Gly Th - #r Asn Pro Gln Gly Glu         #           270                                                               - Arg Arg Ala Phe Leu Asp Lys Gly Thr Tyr Th - #r Leu Ser Trp Lys Leu         #       285                                                                   - Glu Asn Arg Thr Ala Tyr Cys Pro Leu Gln Hi - #s Trp Gln Thr Phe Asp         #   300                                                                       - Ser Thr Ile Ala Thr Glu Thr Gly Lys Ser Il - #e His Phe Val Thr Asp         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Gly Thr Ser Ser Phe Val Thr Asn Thr Th - #r Val Gly Ile Glu Leu         #               335                                                           - Pro Asp Ala Phe Lys Cys Ile Glu Glu Gln Va - #l Asn Lys Thr Met His         #           350                                                               - Glu Lys Tyr Glu Ala Val Gln Asp Arg Tyr Th - #r Lys Gly Gln Glu Ala         #       365                                                                   - Ile Thr Tyr Phe Ile Thr Ser Gly Gly Leu Le - #u Leu Ala Trp Leu Pro         #   380                                                                       - Leu Thr Pro Arg Ser Leu Ala Thr Val Lys As - #n Leu Thr Glu Leu Thr         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Thr Pro Thr Ser Ser Pro Pro Ser Ser Pro Se - #r Pro Pro Ala Pro Ser         #               415                                                           - Ala Ala Arg Gly Ser Thr Pro Ala Ala Val Le - #u Arg Arg Arg Arg Arg         #           430                                                               - Asp Ala Gly Asn Ala Thr Thr Pro Val Pro Pr - #o Thr Ala Pro Gly Lys         #       445                                                                   - Ser Leu Gly Thr Leu Asn Asn Pro Ala Thr Va - #l Gln Ile Gln Phe Ala         #   460                                                                       - Tyr Asp Ser Leu Arg Arg Gln Ile Asn Arg Me - #t Leu Gly Asp Leu Ala         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Arg Ala Trp Cys Leu Glu Gln Lys Arg Gln As - #n Met Val Leu Arg Glu         #               495                                                           - Leu Thr Lys Ile Asn Pro Thr Thr Val Met Se - #r Ser Ile Tyr Gly Lys         #           510                                                               - Ala Val Ala Ala Lys Arg Leu Gly Asp Val Il - #e Ser Val Ser Gln Cys         #       525                                                                   - Val Pro Val Asn Gln Ala Thr Val Thr Leu Ar - #g Lys Ser Met Arg Val         #   540                                                                       - Pro Gly Ser Glu Thr Met Cys Tyr Ser Arg Pr - #o Leu Val Ser Phe Ser         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Phe Ile Asn Asp Thr Lys Thr Tyr Glu Gly Gl - #n Leu Gly Thr Asp Asn         #               575                                                           - Glu Ile Phe Leu Thr Lys Lys Met Thr Glu Va - #l Cys Gln Ala Thr Ser         #           590                                                               - Gln Tyr Tyr Phe Gln Ser Gly Asn Glu Ile Hi - #s Val Tyr Asn Asp Tyr         #       605                                                                   - His His Phe Lys Thr Ile Glu Leu Asp Gly Il - #e Ala Thr Leu Gln Thr         #   620                                                                       - Phe Ile Ser Leu Asn Thr Ser Leu Ile Glu As - #n Ile Asp Phe Ala Ser         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Leu Glu Leu Tyr Ser Arg Asp Glu Gln Arg Al - #a Ser Asn Val Phe Asp         #               655                                                           - Leu Glu Gly Ile Phe Arg Glu Tyr Asn Phe Gl - #n Ala Gln Asn Ile Ala         #           670                                                               - Gly Leu Arg Lys Asp Leu Asp Asn Ala Val Se - #r Asn Gly Arg Asn Gln         #       685                                                                   - Phe Val Asp Gly Leu Gly Glu Leu Met Asp Se - #r Leu Gly Ser Val Gly         #   700                                                                       - Gln Ser Ile Thr Asn Leu Val Ser Thr Val Gl - #y Gly Leu Phe Ser Ser         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Leu Val Ser Gly Phe Ile Ser Phe Phe Lys As - #n Pro Phe Gly Gly Met         #               735                                                           - Leu Ile Leu Val Leu Val Ala Gly Val Val Il - #e Leu Val Ile Ser Leu         #           750                                                               - Thr Arg Arg Thr Arg Gln Met Ser Gln Gln Pr - #o Val Gln Met Leu Tyr         #       765                                                                   - Pro Gly Ile Asp Glu Leu Ala Gln Gln His Al - #a Ser Gly Glu Gly Pro         #   780                                                                       - Gly Ile Asn Pro Ile Ser Lys Thr Glu Leu Gl - #n Ala Ile Met Leu Ala         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Leu His Glu Gln Asn Gln Glu Gln Lys Arg Al - #a Ala Gln Arg Ala Ala         #               815                                                           - Gly Pro Ser Val Ala Ser Arg Ala Leu Gln Al - #a Ala Arg Asp Arg Phe         #           830                                                               - Pro Gly Leu Arg Arg Arg Arg Tyr His Asp Pr - #o Glu Thr Ala Ala Ala         #       845                                                                   - Leu Leu Gly Glu Ala Glu Thr Glu Phe                                         #   855                                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2280 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - CGAGCCCTAA TTATTGGTTT GTATATGACT GTTGGAATTT GTTACATTTT TA - #TTAAAACA         60                                                                          - ATAAATTAAA TTTTTTAAAC TATATTACGG TTGTGTGTGT TTTAAGTTTT AA - #ATAAAGCA        120                                                                          - ATATTTCGAA TTCACATTTA TCAAAAACAT TAAAACCCAA CACAAAAAAA TT - #TCTATAAT        180                                                                          - CATTAAGGTA ATAAGTCAAA ATGAGTTTTA AAAATTTTTA TCTAATATAT GT - #AATTATAA        240                                                                          - TTTTTATAAA CTCGATAATA ACTTCGGCAT CTACATCCAA ACCTTCAACA CC - #TACCATAA        300                                                                          - TTCCAACTTC AGCAAATGAA TCACCTGCTT CCATAGATAC AACTATAACA AA - #ACCTATAT        360                                                                          - CTACAGAGGC AAATAATTTA AAATCAGTAA GTACCTCAAT TAAACCACCT AA - #AAACTTAA        420                                                                          - AAAAAAAATT ACTTAAATCT AAATGTAGAG ATAATGTTAT TTATAGGCCA TA - #TTTTAGTC        480                                                                          - AATTAGAAAT TAACTGTACT ATAACTAAAA AGCAAAATTT AAGTAATCCT TT - #AATTGAGT        540                                                                          - TATGGTTTAA AGAACTTTCT ACATATAATA AAACCAATGA AAATGTTGAA AG - #TTTAAAAA        600                                                                          - CAGATATATC AAAAAATATT TTATTATTTT CGACAAAAAA TAATAGTGAT AA - #CTTTTATA        660                                                                          - ATGATTTTTT ATTAGGTATA CAAAATCAAC CAGTAAATTA TAAACTTTAC GG - #TTCCCAAT        720                                                                          - TTTATGATAA TGGAAACATA TTACTAAATA TAAAGTCGGT TGACTTTAAA AC - #CTCTGGAA        780                                                                          - TATATACTTG GAAACTATAT AATTCAAATA ATGAAAGTAT TTTTGAAACT TT - #TAAAATTC        840                                                                          - AAGTATATGC ATATCATTCC CCAAATGTAA ACTTAAAATC AAACCCAAGT TT - #ATATAATG        900                                                                          - AAAACTACAG CGCTATTTGT ACAATAGCAA ATTACTTTCC ATTGGAATCT AC - #GGAAATAT        960                                                                          - TTTGGTTTAA CGATGGACAA CCTATTGATA AAAAATATAT AGATGAAACT TA - #TAGTGTAT       1020                                                                          - GGATTGACGG TCTTATAACA CGCACTTCAA TATTATCCCT TCCCTTTTCC GA - #AGCCATGG       1080                                                                          - AAAGCCCCCC CAATTTGCGA TGTAATGTTG AATGGTATAA AAATTCAAAG GC - #ATCAAAAA       1140                                                                          - AATTTTCAAA TACCGTTATT CCAAAAGTTT ACTATAAACC TTTTATATCT AT - #AAAATTTG       1200                                                                          - ATAATGGTTT AGCTATTTGT GATGCTAAAT GTGTTTCCCG TGAAAATAAT AA - #ATTACAAT       1260                                                                          - GGTTAGTTAA AGATATACCT ATAAATGGTG ATGATATTAT AAGCGGCCCC TG - #TTTAAACC       1320                                                                          - ACCCTGGTTT GGTCAATATT CAAAATAAAA TAGATATATC GGATTATGAT GA - #ACCTGTTA       1380                                                                          - CCTATAAATG TTCAATTATT GGTTATCCAA TAATTTTTCC CAACTTTTAT GA - #TGAAAAGG       1440                                                                          - TGTTTGATGC ATCGGATGAA AATGTTAGTA AATCGATGTT AATAAGTATT AC - #CACAATAA       1500                                                                          - TTGGTGGAGC CATTTTTGTT ATAGTATTGA TTTTTATAAC AGCTTTATGT TT - #TTATTGTT       1560                                                                          - CAAAAAATAA TAAGATCTAA TATCAATATT TACGTAAATG GATTATATAA TG - #TTATATTC       1620                                                                          - GTGTTATTAT GATTTATAAG TTCATCAAAT TTAAAAATTT GTATAGTATT AA - #GATTTTTA       1680                                                                          - ATAGGGGTAT CGTTTAATAT GGCTCAGTTA GTTTTAACTG ATATTCCCCT CG - #AAGATGTG       1740                                                                          - GAAAATAAAA ATACTTCATC CGACGAAGAA ACAACTAACT TAAACCAGAA AA - #AATCAACA       1800                                                                          - TGTCAATGTT TATGTGTTAC CCTTGGATTT TTTGCAGCTG GAATTTTATT AA - #CCATAGCT       1860                                                                          - GCAATAATTT TTACTTTTAT TTTTACAGTA CCATTAGAAA TGCTTGGATC TA - #TTAATTGT       1920                                                                          - CCTCCATCTA CATTTGGTAT TGATAATGTT TGTATCGAAC CAATAAAAAA AT - #CTATTAAT       1980                                                                          - TCTTATTCAG AATTATCTAA AATATGTTAT GATAGATTGT CAAATCCGAT AA - #ATCAGAGT       2040                                                                          - ACTATTAACT CCTTATTAAC TGTTTTAAAT ATGTTTGCAG ATAAAAACTA TG - #AAAATGTT       2100                                                                          - TATAATTGTA ATACAATGAG TGAAAAAACA TGTAATTCAT CAATAGCTAT TT - #GTCAAACT       2160                                                                          - AATCATCCAC TAAGTTCATT GGGAAATTTT GTTATTAAAA TTAGAAAAAT TT - #TTGGGTTT       2220                                                                          - AAATAATAAA TAAAATAAAT AAACATTACT TTTTGTTTTT GTCTTTATTA AA - #CAGTTGTA       2280                                                                          - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 459 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Met Ser Phe Lys Asn Phe Tyr Leu Ile Tyr Va - #l Ile Ile Ile Phe Ile         #                15                                                           - Asn Ser Ile Ile Thr Ser Ala Ser Thr Ser Ly - #s Pro Ser Thr Pro Thr         #            30                                                               - Ile Ile Pro Thr Ser Ala Asn Glu Ser Pro Al - #a Ser Ile Asp Thr Thr         #        45                                                                   - Ile Thr Lys Pro Ile Ser Thr Glu Ala Asn As - #n Leu Lys Ser Val Ser         #    60                                                                       - Thr Ser Ile Lys Pro Pro Lys Asn Leu Lys Ly - #s Lys Leu Leu Lys Ser         #80                                                                           - Lys Cys Arg Asp Asn Val Ile Tyr Arg Pro Ty - #r Phe Ser Gln Leu Glu         #                95                                                           - Ile Asn Cys Thr Ile Thr Lys Lys Gln Asn Le - #u Ser Asn Pro Leu Ile         #           110                                                               - Glu Leu Trp Phe Lys Glu Leu Ser Thr Tyr As - #n Lys Thr Asn Glu Asn         #       125                                                                   - Val Glu Ser Leu Lys Thr Asp Ile Ser Lys As - #n Ile Leu Leu Phe Ser         #   140                                                                       - Thr Lys Asn Asn Ser Asp Asn Phe Tyr Asn As - #p Phe Leu Leu Gly Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Asn Gln Pro Val Asn Tyr Lys Leu Tyr Gl - #y Ser Gln Phe Tyr Asp         #               175                                                           - Asn Gly Asn Ile Leu Leu Asn Ile Lys Ser Va - #l Asp Phe Lys Thr Ser         #           190                                                               - Gly Ile Tyr Thr Trp Lys Leu Tyr Asn Ser As - #n Asn Glu Ser Ile Phe         #       205                                                                   - Glu Thr Phe Lys Ile Gln Val Tyr Ala Tyr Hi - #s Ser Pro Asn Val Asn         #   220                                                                       - Leu Lys Ser Asn Pro Ser Leu Tyr Asn Glu As - #n Tyr Ser Ala Ile Cys         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Ile Ala Asn Tyr Phe Pro Leu Glu Ser Th - #r Glu Ile Phe Trp Phe         #               255                                                           - Asn Asp Gly Gln Pro Ile Asp Lys Lys Tyr Il - #e Asp Glu Thr Tyr Ser         #           270                                                               - Val Trp Ile Asp Gly Leu Ile Thr Arg Thr Se - #r Ile Leu Ser Leu Pro         #       285                                                                   - Phe Ser Glu Ala Met Glu Ser Pro Pro Asn Le - #u Arg Cys Asn Val Glu         #   300                                                                       - Trp Tyr Lys Asn Ser Lys Ala Ser Lys Lys Ph - #e Ser Asn Thr Val Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Lys Val Tyr Tyr Lys Pro Phe Ile Ser Il - #e Lys Phe Asp Asn Gly         #               335                                                           - Leu Ala Ile Cys Asp Ala Lys Cys Val Ser Ar - #g Glu Asn Asn Lys Leu         #           350                                                               - Gln Trp Leu Val Lys Asp Ile Pro Ile Asn Gl - #y Asp Asp Ile Ile Ser         #       365                                                                   - Gly Pro Cys Leu Asn His Pro Gly Leu Val As - #n Ile Gln Asn Lys Ile         #   380                                                                       - Asp Ile Ser Asp Tyr Asp Glu Pro Val Thr Ty - #r Lys Cys Ser Ile Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Tyr Pro Ile Ile Phe Pro Asn Phe Tyr As - #p Glu Lys Val Phe Asp         #               415                                                           - Ala Ser Asp Glu Asn Val Ser Lys Ser Met Le - #u Ile Ser Ile Thr Thr         #           430                                                               - Ile Ile Gly Gly Ala Ile Phe Val Ile Val Le - #u Ile Phe Ile Thr Ala         #       445                                                                   - Leu Cys Phe Tyr Cys Ser Lys Asn Asn Lys Il - #e                             #   455                                                                       - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 175 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Met Ala Gln Leu Val Leu Thr Asp Ile Pro Le - #u Glu Asp Val Glu Asn         #                15                                                           - Lys Asn Thr Ser Ser Asp Glu Glu Thr Thr As - #n Leu Asn Gln Lys Lys         #            30                                                               - Ser Thr Cys Gln Cys Leu Cys Val Thr Leu Gl - #y Phe Phe Ala Ala Gly         #        45                                                                   - Ile Leu Leu Thr Ile Ala Ala Ile Ile Phe Th - #r Phe Ile Phe Thr Val         #    60                                                                       - Pro Leu Glu Met Leu Gly Ser Ile Asn Cys Pr - #o Pro Ser Thr Phe Gly         #80                                                                           - Ile Asp Asn Val Cys Ile Glu Pro Ile Lys Ly - #s Ser Ile Asn Ser Tyr         #                95                                                           - Ser Glu Leu Ser Lys Ile Cys Tyr Asp Arg Le - #u Ser Asn Pro Ile Asn         #           110                                                               - Gln Ser Thr Ile Asn Ser Leu Leu Thr Val Le - #u Asn Met Phe Ala Asp         #       125                                                                   - Lys Asn Tyr Glu Asn Val Tyr Asn Cys Asn Th - #r Met Ser Glu Lys Thr         #   140                                                                       - Cys Asn Ser Ser Ile Ala Ile Cys Gln Thr As - #n His Pro Leu Ser Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Gly Asn Phe Val Ile Lys Ile Arg Lys Il - #e Phe Gly Phe Lys             #               175                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 459 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Met Ser Phe Lys Asn Phe Tyr Leu Ile Tyr Va - #l Ile Ile Ile Phe Ile         #                15                                                           - Asn Ser Ile Ile Thr Ser Ala Ser Thr Ser Ly - #s Pro Ser Thr Pro Thr         #            30                                                               - Ile Ile Pro Thr Ser Ala Asn Glu Ser Pro Al - #a Ser Ile Asp Thr Thr         #        45                                                                   - Ile Thr Lys Pro Ile Ser Thr Glu Ala Asn As - #n Leu Lys Ser Val Ser         #    60                                                                       - Thr Ser Ile Lys Pro Pro Lys Asn Leu Lys Ly - #s Lys Leu Leu Lys Ser         #80                                                                           - Lys Cys Arg Asp Asn Val Ile Tyr Arg Pro Ty - #r Phe Ser Gln Leu Glu         #                95                                                           - Ile Asn Cys Thr Ile Thr Lys Lys Gln Asn Le - #u Ser Asn Pro Leu Ile         #           110                                                               - Glu Leu Trp Phe Lys Glu Leu Ser Thr Tyr As - #n Lys Thr Asn Glu Asn         #       125                                                                   - Val Glu Ser Leu Lys Thr Asp Ile Ser Lys As - #n Ile Leu Leu Phe Ser         #   140                                                                       - Thr Lys Asn Asn Ser Asp Asn Phe Tyr Asn As - #p Phe Leu Leu Gly Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Asn Gln Pro Val Asn Tyr Lys Leu Tyr Gl - #y Ser Gln Phe Tyr Asp         #               175                                                           - Asn Gly Asn Ile Leu Leu Asn Ile Lys Ser Va - #l Asp Phe Lys Thr Ser         #           190                                                               - Gly Ile Tyr Thr Trp Lys Leu Tyr Asn Ser As - #n Asn Glu Ser Ile Phe         #       205                                                                   - Glu Thr Phe Lys Ile Gln Val Tyr Ala Tyr Hi - #s Ser Pro Asn Val Asn         #   220                                                                       - Leu Lys Ser Asn Pro Ser Leu Tyr Asn Glu As - #n Tyr Ser Ala Ile Cys         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Ile Ala Asn Tyr Phe Pro Leu Glu Ser Th - #r Glu Ile Phe Trp Phe         #               255                                                           - Asn Asp Gly Gln Pro Ile Asp Lys Lys Tyr Il - #e Asp Glu Thr Tyr Ser         #           270                                                               - Val Trp Ile Asp Gly Leu Ile Thr Arg Thr Se - #r Ile Leu Ser Leu Pro         #       285                                                                   - Phe Ser Glu Ala Met Glu Ser Pro Pro Asn Le - #u Arg Cys Asn Val Glu         #   300                                                                       - Trp Tyr Lys Asn Ser Lys Ala Ser Lys Lys Ph - #e Ser Asn Thr Val Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Lys Val Tyr Tyr Lys Pro Phe Ile Ser Il - #e Lys Phe Asp Asn Gly         #               335                                                           - Leu Ala Ile Cys Asp Ala Lys Cys Val Ser Ar - #g Glu Asn Asn Lys Leu         #           350                                                               - Gln Trp Leu Val Lys Asp Ile Pro Ile Asn Gl - #y Asp Asp Ile Ile Ser         #       365                                                                   - Gly Pro Cys Leu Asn His Pro Gly Leu Val As - #n Ile Gln Asn Lys Ile         #   380                                                                       - Asp Ile Ser Asp Tyr Asp Glu Pro Val Thr Ty - #r Lys Cys Ser Ile Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Tyr Pro Ile Ile Phe Pro Asn Phe Tyr As - #p Glu Lys Val Phe Asp         #               415                                                           - Ala Ser Asp Glu Asn Val Ser Lys Ser Met Le - #u Ile Ser Ile Thr Thr         #           430                                                               - Ile Ile Gly Gly Ala Ile Phe Val Ile Val Le - #u Ile Phe Ile Thr Ala         #       445                                                                   - Leu Cys Phe Tyr Cys Ser Lys Asn Asn Lys Il - #e                             #   455                                                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 533 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Met Arg Arg Tyr Arg Met Gly Arg Gly Ile Ty - #r Leu Leu Tyr Ile Cys         #                15                                                           - Leu Leu Tyr Thr Tyr Leu Gln Phe Gly Thr Se - #r Ser Thr Thr Ala Val         #            30                                                               - Ser Ile Glu Asn Ser Asp Asn Ser Thr Ala Gl - #u Met Leu Ser Ser Thr         #        45                                                                   - Ser Met Ser Ala Thr Thr Pro Ile Ser Gln Pr - #o Thr Ser Pro Phe Thr         #    60                                                                       - Thr Pro Thr Arg Arg Ser Thr Asn Ile Ala Th - #r Ser Ser Ser Thr Thr         #80                                                                           - Gln Ala Ser Gln Pro Thr Ser Thr Leu Thr Th - #r Leu Thr Arg Ser Ser         #                95                                                           - Thr Thr Ile Ala Thr Ser Pro Ser Thr Thr Gl - #n Ala Ala Thr Phe Ile         #           110                                                               - Gly Ser Ser Thr Asp Ser Asn Thr Thr Leu Le - #u Lys Thr Thr Lys Lys         #       125                                                                   - Pro Lys Arg Lys Lys Asn Lys Asn Asn Gly Al - #a Arg Phe Lys Leu Asp         #   140                                                                       - Cys Gly Tyr Lys Gly Val Ile Tyr Arg Pro Ty - #r Phe Ser Pro Leu Gln         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Asn Cys Thr Leu Pro Thr Glu Pro His Il - #e Thr Asn Pro Ile Asp         #               175                                                           - Phe Glu Ile Trp Phe Lys Pro Arg Thr Arg Ph - #e Gly Asp Phe Leu Gly         #           190                                                               - Asp Lys Glu Asp Phe Val Gly Asn His Thr Ar - #g Thr Ser Ile Leu Leu         #       205                                                                   - Phe Ser Ser Arg Asn Gly Ser Val Asn Ser Me - #t Asp Leu Gly Asp Ala         #   220                                                                       - Thr Leu Gly Ile Leu Gln Ser Arg Ile Pro As - #p Tyr Thr Leu Tyr Asn         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Pro Ile Gln His Thr Glu Ala Met Ser Le - #u Gly Ile Lys Ser Val         #               255                                                           - Glu Ser Ala Thr Ser Gly Val Tyr Thr Trp Ar - #g Val Tyr Gly Gly Asp         #           270                                                               - Gly Leu Asn Lys Thr Val Leu Gly Gln Val As - #n Val Ser Val Val Ala         #       285                                                                   - Tyr His Pro Pro Ser Val Asn Leu Thr Pro Ar - #g Ala Ser Leu Phe Asn         #   300                                                                       - Lys Thr Phe Glu Ala Val Cys Ala Val Ala As - #n Tyr Phe Pro Arg Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Thr Lys Leu Thr Trp Tyr Leu Asp Gly Lys Pr - #o Ile Glu Arg Gln Tyr         #               335                                                           - Ile Ser Asp Thr Ala Ser Val Trp Ile Asp Gl - #y Leu Ile Thr Arg Ser         #           350                                                               - Ser Val Leu Ala Ile Pro Thr Thr Glu Thr As - #p Ser Glu Lys Pro Asp         #       365                                                                   - Ile Arg Cys Asp Leu Glu Trp His Glu Ser Pr - #o Val Ser Tyr Lys Arg         #   380                                                                       - Phe Thr Lys Ser Val Ala Pro Asp Val Tyr Ty - #r Pro Pro Thr Val Ser         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Thr Phe Ala Asp Thr Arg Ala Ile Cys As - #p Val Lys Cys Val Pro         #               415                                                           - Arg Asp Gly Ile Ser Leu Met Trp Lys Ile Gl - #y Asn Tyr His Leu Pro         #           430                                                               - Lys Ala Met Ser Ala Asp Ile Leu Ile Thr Gl - #y Pro Cys Ile Glu Arg         #       445                                                                   - Pro Gly Leu Val Asn Ile Gln Ser Met Cys As - #p Ile Ser Glu Thr Asp         #   460                                                                       - Gly Pro Val Ser Tyr Thr Cys Gln Thr Ile Gl - #y Tyr Pro Pro Ile Leu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Pro Gly Phe Tyr Asp Thr Gln Val Tyr Asp Al - #a Ser Pro Glu Ile Val         #               495                                                           - Ser Glu Ser Met Leu Val Ser Val Val Ala Va - #l Ile Leu Gly Ala Val         #           510                                                               - Leu Ile Thr Val Phe Ile Phe Ile Thr Ala Le - #u Cys Leu Tyr Tyr Ser         #       525                                                                   - His Pro Arg Arg Leu                                                             530                                                                       - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 468 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Met Trp Leu Pro Asn Leu Val Arg Phe Val Al - #a Val Ala Tyr Leu Ile         #                15                                                           - Cys Ala Gly Ala Ile Leu Thr Tyr Ala Ser Gl - #y Ala Ser Ala Ser Ser         #            30                                                               - Ser Gln Ser Thr Pro Ala Thr Pro Thr His Th - #r Thr Pro Asn Leu Thr         #        45                                                                   - Thr Ala His Gly Ala Gly Ser Asp Asn Thr Th - #r Asn Ala Asn Gly Thr         #    60                                                                       - Glu Ser Thr His Ser His Glu Thr Thr Ile Th - #r Cys Thr Lys Ser Leu         #80                                                                           - Ile Ser Val Pro Tyr Tyr Lys Ser Val Asp Me - #t Asn Cys Thr Thr Ser         #                95                                                           - Val Gly Val Asn Tyr Ser Glu Tyr Arg Leu Gl - #u Ile Tyr Leu Asn Gln         #           110                                                               - Arg Thr Pro Phe Ser Gly Thr Pro Pro Gly As - #p Glu Glu Asn Tyr Ile         #       125                                                                   - Asn His Asn Ala Thr Lys Asp Gln Thr Leu Le - #u Leu Phe Ser Thr Ala         #   140                                                                       - Glu Arg Lys Lys Ser Arg Arg Gly Asp Leu Se - #r Val His Pro Ser Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Gly Glu Asn Tyr Arg Ala Thr Cys Val Va - #l Ala Ser Tyr Phe Pro         #               175                                                           - His Ser Ser Val Lys Leu Arg Trp Tyr Lys As - #n Ala Arg Glu Val Asp         #           190                                                               - Phe Thr Lys Tyr Val Thr Asn Ala Ser Ser Va - #l Trp Val Asp Gly Leu         #       205                                                                   - Ile Thr Arg Ile Ser Thr Val Ser Ile Pro Va - #l Asp Pro Glu Glu Glu         #   220                                                                       - Tyr Thr Gly Gln Leu Gly Val Ile Pro Asp Ar - #g Leu Pro Lys Arg Gln         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Phe Asn Leu Pro Leu His Thr Glu Gly Gl - #y Thr Lys Phe Pro Leu         #               255                                                           - Thr Ile Lys Ser Val Asp Trp Arg Thr Ala Gl - #y Ile Tyr Val Trp Ser         #           270                                                               - Leu Tyr Ala Lys Asn Gly Thr Leu Val Asn Se - #r Thr Ser Val Thr Val         #       285                                                                   - Ser Thr Tyr Asn Ala Pro Leu Leu Pro Ser Le - #u Arg Cys Ser Ile Asp         #   300                                                                       - Trp Tyr Arg Asp Glu Val Ser Phe Ala Arg Il - #e Ala Lys Ala Gly Thr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Ser Val Phe Val Ala Pro Thr Val Ser Va - #l Ser Val Glu Asp Gly         #               335                                                           - Asp Ala Val Cys Thr Ala Lys Cys Val Pro Se - #r Thr Gly Val Phe Val         #           350                                                               - Ser Trp Ser Val Asn Asp His Leu Pro Gly Va - #l Pro Ser Gln Asp Met         #       365                                                                   - Thr Thr Gly Val Cys Pro Ser His Ser Gly Le - #u Val Asn Met Gln Ser         #   380                                                                       - Arg Arg Pro Leu Ser Glu Glu Asn Gly Glu Ar - #g Glu Tyr Ser Cys Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ile Glu Gly Tyr Pro Asp Gly Leu Pro Met Ph - #e Ser Asp Thr Val Val         #               415                                                           - Tyr Asp Ala Ser Pro Ile Val Glu Asp Arg Pr - #o Val Leu Thr Ser Ile         #           430                                                               - Ile Ala Val Thr Cys Gly Ala Ala Ala Leu Al - #a Leu Val Val Leu Ile         #       445                                                                   - Thr Ala Val Cys Phe Tyr Cys Ser Lys Pro Se - #r Gln Ala Pro Tyr Lys         #   460                                                                       - Lys Ser Asp Phe                                                             465                                                                           - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 511 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Met Ala Pro Gly Arg Val Gly Leu Ala Val Va - #l Leu Trp Ser Leu Leu         #                15                                                           - Trp Leu Gly Ala Gly Val Ser Gly Gly Ser Gl - #u Thr Ala Ser Thr Gly         #            30                                                               - Pro Thr Ile Thr Ala Gly Ala Val Thr Asn Al - #a Ser Glu Ala Pro Thr         #        45                                                                   - Ser Gly Ser Pro Gly Ser Ala Ala Ser Pro Gl - #u Val Thr Pro Thr Ser         #    60                                                                       - Thr Pro Asn Pro Asn Asn Val Thr Gln Asn Ly - #s Thr Thr Pro Thr Glu         #80                                                                           - Pro Ala Ser Pro Pro Thr Thr Pro Lys Pro Th - #r Ser Thr Pro Lys Ser         #                95                                                           - Pro Pro Thr Ser Thr Pro Asp Pro Lys Pro Ly - #s Asn Asn Thr Thr Pro         #           110                                                               - Ala Lys Ser Gly Arg Pro Thr Lys Pro Pro Gl - #y Pro Val Trp Cys Asp         #       125                                                                   - Arg Arg Asp Pro Leu Ala Arg Tyr Gly Ser Ar - #g Val Gln Ile Arg Cys         #   140                                                                       - Arg Phe Arg Asn Ser Thr Arg Met Glu Phe Ar - #g Leu Gln Ile Trp Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Ser Met Gly Pro Ser Pro Pro Ile Ala Pr - #o Ala Pro Asp Leu Glu         #               175                                                           - Glu Val Leu Thr Asn Ile Thr Ala Pro Pro Gl - #y Gly Leu Leu Val Tyr         #           190                                                               - Asp Ser Ala Pro Asn Leu Thr Asp Pro His Va - #l Leu Trp Ala Glu Gly         #       205                                                                   - Ala Gly Pro Gly Ala Asp Pro Pro Leu Tyr Se - #r Val Thr Gly Pro Leu         #   220                                                                       - Pro Thr Gln Arg Leu Ile Ile Gly Glu Val Th - #r Pro Ala Thr Gln Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Tyr Tyr Leu Ala Trp Gly Arg Met Asp Se - #r Pro His Glu Tyr Gly         #               255                                                           - Thr Trp Val Arg Val Arg Met Phe Arg Pro Pr - #o Ser Leu Thr Leu Gln         #           270                                                               - Pro His Ala Val Met Glu Gly Gln Pro Phe Ly - #s Ala Thr Cys Thr Ala         #       285                                                                   - Ala Ala Tyr Tyr Pro Arg Asn Pro Val Glu Ph - #e Val Trp Phe Glu Asp         #   300                                                                       - Asp His Gln Val Phe Asn Pro Gly Gln Ile As - #p Thr Gln Thr His Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - His Pro Asp Gly Phe Thr Thr Val Ser Thr Va - #l Thr Ser Glu Ala Val         #               335                                                           - Gly Gly Gln Val Pro Pro Arg Thr Phe Thr Cy - #s Gln Met Thr Trp His         #           350                                                               - Arg Asp Ser Val Thr Phe Ser Arg Arg Asn Al - #a Thr Gly Leu Ala Leu         #       365                                                                   - Val Leu Pro Arg Pro Thr Ile Thr Met Glu Ph - #e Gly Val Arg Ile Val         #   380                                                                       - Val Cys Thr Ala Gly Cys Val Pro Glu Gly Va - #l Thr Phe Ala Trp Phe         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Gly Asp Asp Pro Ser Pro Ala Ala Lys Se - #r Ala Val Thr Ala Gln         #               415                                                           - Glu Ser Cys Asp His Pro Gly Leu Ala Thr Va - #l Arg Ser Thr Leu Pro         #           430                                                               - Ile Ser Tyr Asp Tyr Ser Glu Tyr Ile Cys Ar - #g Leu Thr Gly Tyr Pro         #       445                                                                   - Ala Gly Ile Pro Val Leu Glu His His Gly Se - #r His Gln Pro Pro Pro         #   460                                                                       - Arg Asp Pro Thr Glu Arg Gln Val Ile Glu Al - #a Ile Glu Trp Val Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ile Gly Ile Gly Val Leu Ala Ala Gly Val Le - #u Val Val Thr Ala Ile         #               495                                                           - Val Tyr Val Val Arg Thr Ser Gln Ser Arg Gl - #n Arg His Arg Arg             #           510                                                               - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1320 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - GATATTTAAT AAAACTATTA TGAAACTTCT TATAACTTAT TTGTTTTTAT TA - #AATGGGTT         60                                                                          - GGGTTGGTTT TAAAATTACA TACGTGTATT AAGAATTAAC ATCATAAAGG AC - #ACACCCAT        120                                                                          - GAAAAACATT TAAATTCTAT TAATTTGAAC GGATTAAACA TTTTCTCATT TT - #AAGAGTTG        180                                                                          - CTACGACTTT TGATAGTAAA ATGATTAAAC TTCTATTTAT CTTATTTTAT TT - #TAACCCAA        240                                                                          - TAACTGGATA TAAATGGGTA GACCCTCCTC GTAGGTATAA TTACACCGTT TT - #AAGAATGA        300                                                                          - TTCCAGATAT TCCAAATCCA ATGGATCCTT CTAAAAACGC TGAAGTTCGG TA - #TGTAACTT        360                                                                          - CTACTGACCC ATGTGATATG GTTGCTTTGA TTTCTAATCC AAATATAGAA TC - #TACAATTA        420                                                                          - AAACGATTCA ATTTGTGCAA AAGAAAAAAT TTTACAATGC ATCTCTTAGT TG - #GTTTAAAG        480                                                                          - TTGGAGATGA TTGTACATAT CCAATATATT TAATTCAATA TTTTGATTGT GA - #TCCTCAAA        540                                                                          - GAGAATTTGG CATATGTTTA AAAAGATCTC CAGATTTTTG GAAACCATCG TT - #AGTTGGTT        600                                                                          - ACACATTTTT AACTGATGAT GAATTGGGAT TAGTTTTAGC TGCCCCCGCT CC - #ATTTAATC        660                                                                          - AAGGTCAATA TAGACGGGTT ATTCAAATTG AAAATGAAGT TTTTTATACT GA - #TTTTATGG        720                                                                          - TTCAATTACC ACGAGAAACT TGTTATTTTT CTAAAGAAGA TAAATTTGAA CC - #AACTTTTA        780                                                                          - TGGAATGGTG TAAGGAATCT AGATCTGTAG GAGCATCAAA AGTTGACGAT GA - #ACTTTTTT        840                                                                          - ATCTAAATAG AGCTGGTCCC CAAACCCTGC TTAAATATTA TGTTATTAAA GA - #TTTTTATA        900                                                                          - GACTTAACGG TAGAGAACCT CCAATAAAAT TTAAAGAAGC TCTTAGATAC GA - #TATACCAT        960                                                                          - ATAAAGTGAA TGATAAATTT GATGATGAAT TACCATCGAG GCCACATATT AG - #TAATACTA       1020                                                                          - TTAATAAAAC TATTAAAGAA ATTGTAAATC TTGAAGATTA TTTTAAAAAT AC - #AAATGTTA       1080                                                                          - TAGATACTAC TACCCCAACA CCAATAAATA ATACCCCAAA AAATATAACC GT - #GGGAATTG       1140                                                                          - TTATAATTAT ATTAATAATA CTATTTATAA TTGGATTTTT TGTTTATAAA AG - #ACAAAAAA       1200                                                                          - TATATAATAA TTATAAAAAA TTAACAACAA ATGTTTAGCC TTTATAAATT AA - #TTTACAGA       1260                                                                          - ATAAACAACT GGGCGGTCTT TTGTTTAATA AAAATTCATG TACCTACAAC TT - #TTATTCAC       1320                                                                          - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 345 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Met Ile Lys Leu Leu Phe Ile Leu Phe Tyr Ph - #e Asn Pro Ile Thr Gly         #                15                                                           - Tyr Lys Trp Val Asp Pro Pro Arg Arg Tyr As - #n Tyr Thr Val Leu Arg         #            30                                                               - Met Ile Pro Asp Ile Pro Asn Pro Met Asp Pr - #o Ser Lys Asn Ala Glu         #        45                                                                   - Val Arg Tyr Val Thr Ser Thr Asp Pro Cys As - #p Met Val Ala Leu Ile         #    60                                                                       - Ser Asn Pro Asn Ile Glu Ser Thr Ile Lys Th - #r Ile Gln Phe Val Gln         #80                                                                           - Lys Lys Lys Phe Tyr Asn Ala Ser Leu Ser Tr - #p Phe Lys Val Gly Asp         #                95                                                           - Asp Cys Thr Tyr Pro Ile Tyr Leu Ile Gln Ty - #r Phe Asp Cys Asp Pro         #           110                                                               - Gln Arg Glu Phe Gly Ile Cys Leu Lys Arg Se - #r Pro Asp Phe Trp Lys         #       125                                                                   - Pro Ser Leu Val Gly Tyr Thr Phe Leu Thr As - #p Asp Glu Leu Gly Leu         #   140                                                                       - Val Leu Ala Ala Pro Ala Pro Phe Asn Gln Gl - #y Gln Tyr Arg Arg Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Gln Ile Glu Asn Glu Val Phe Tyr Thr As - #p Phe Met Val Gln Leu         #               175                                                           - Pro Arg Glu Thr Cys Tyr Phe Ser Lys Glu As - #p Lys Phe Glu Pro Thr         #           190                                                               - Phe Met Glu Trp Cys Lys Glu Ser Arg Ser Va - #l Gly Ala Ser Lys Val         #       205                                                                   - Asp Asp Glu Leu Phe Tyr Leu Asn Arg Ala Gl - #y Pro Gln Thr Leu Leu         #   220                                                                       - Lys Tyr Tyr Val Ile Lys Asp Phe Tyr Arg Le - #u Asn Gly Arg Glu Pro         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Ile Lys Phe Lys Glu Ala Leu Arg Tyr As - #p Ile Pro Tyr Lys Val         #               255                                                           - Asn Asp Lys Phe Asp Asp Glu Leu Pro Ser Ar - #g Pro His Ile Ser Asn         #           270                                                               - Thr Ile Asn Lys Thr Ile Lys Glu Ile Val As - #n Leu Glu Asp Tyr Phe         #       285                                                                   - Lys Asn Thr Asn Val Ile Asp Thr Thr Thr Pr - #o Thr Pro Ile Asn Asn         #   300                                                                       - Thr Pro Lys Asn Ile Thr Val Gly Ile Val Il - #e Ile Ile Leu Ile Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Phe Ile Ile Gly Phe Phe Val Tyr Lys Ar - #g Gln Lys Ile Tyr Asn         #               335                                                           - Asn Tyr Lys Lys Leu Thr Thr Asn Val                                         #           345                                                               - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 345 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Met Ile Lys Leu Leu Phe Ile Leu Phe Tyr Ph - #e Asn Pro Ile Thr Gly         #                15                                                           - Tyr Lys Trp Val Asp Pro Pro Arg Arg Tyr As - #n Tyr Thr Val Leu Arg         #            30                                                               - Met Ile Pro Asp Ile Pro Asn Pro Met Asp Pr - #o Ser Lys Asn Ala Glu         #        45                                                                   - Val Arg Tyr Val Thr Ser Thr Asp Pro Cys As - #p Met Val Ala Leu Ile         #    60                                                                       - Ser Asn Pro Asn Ile Glu Ser Thr Ile Lys Th - #r Ile Gln Phe Val Gln         #80                                                                           - Lys Lys Lys Phe Tyr Asn Ala Ser Leu Ser Tr - #p Phe Lys Val Gly Asp         #                95                                                           - Asp Cys Thr Tyr Pro Ile Tyr Leu Ile Gln Ty - #r Phe Asp Cys Asp Pro         #           110                                                               - Gln Arg Glu Phe Gly Ile Cys Leu Lys Arg Se - #r Pro Asp Phe Trp Lys         #       125                                                                   - Pro Ser Leu Val Gly Tyr Thr Phe Leu Thr As - #p Asp Glu Leu Gly Leu         #   140                                                                       - Val Leu Ala Ala Pro Ala Pro Phe Asn Gln Gl - #y Gln Tyr Arg Arg Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Gln Ile Glu Asn Glu Val Phe Tyr Thr As - #p Phe Met Val Gln Leu         #               175                                                           - Pro Arg Glu Thr Cys Tyr Phe Ser Lys Glu As - #p Lys Phe Glu Pro Thr         #           190                                                               - Phe Met Glu Trp Cys Lys Glu Ser Arg Ser Va - #l Gly Ala Ser Lys Val         #       205                                                                   - Asp Asp Glu Leu Phe Tyr Leu Asn Arg Ala Gl - #y Pro Gln Thr Leu Leu         #   220                                                                       - Lys Tyr Tyr Val Ile Lys Asp Phe Tyr Arg Le - #u Asn Gly Arg Glu Pro         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Ile Lys Phe Lys Glu Ala Leu Arg Tyr As - #p Ile Pro Tyr Lys Val         #               255                                                           - Asn Asp Lys Phe Asp Asp Glu Leu Pro Ser Ar - #g Pro His Ile Ser Asn         #           270                                                               - Thr Ile Asn Lys Thr Ile Lys Glu Ile Val As - #n Leu Glu Asp Tyr Phe         #       285                                                                   - Lys Asn Thr Asn Val Ile Asp Thr Thr Thr Pr - #o Thr Pro Ile Asn Asn         #   300                                                                       - Thr Pro Lys Asn Ile Thr Val Gly Ile Val Il - #e Ile Ile Leu Ile Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Phe Ile Ile Gly Phe Phe Val Tyr Lys Ar - #g Gln Lys Ile Tyr Asn         #               335                                                           - Asn Tyr Lys Lys Leu Thr Thr Asn Val                                         #           345                                                               - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 374 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Met Met Thr Arg Leu His Phe Trp Trp Cys Gl - #y Ile Phe Ala Val Leu         #                15                                                           - Lys Tyr Leu Val Cys Thr Ser Ser Leu Thr Th - #r Thr Pro Lys Thr Thr         #            30                                                               - Thr Val Tyr Val Lys Gly Phe Asn Ile Pro Pr - #o Leu Arg Tyr Asn Tyr         #        45                                                                   - Thr Gln Ala Arg Ile Val Pro Lys Ile Pro Gl - #n Ala Met Asp Pro Lys         #    60                                                                       - Ile Thr Ala Glu Val Arg Tyr Val Thr Ser Me - #t Asp Ser Cys Gly Met         #80                                                                           - Val Ala Leu Ile Ser Glu Pro Asp Ile Asp Al - #a Thr Ile Arg Thr Ile         #                95                                                           - Gln Leu Ser Gln Lys Lys Thr Tyr Asn Ala Th - #r Ile Ser Trp Phe Lys         #           110                                                               - Val Thr Gln Gly Cys Glu Tyr Pro Met Phe Le - #u Met Asp Met Arg Leu         #       125                                                                   - Cys Asp Pro Lys Arg Glu Phe Gly Ile Cys Al - #a Leu Arg Ser Pro Ser         #   140                                                                       - Tyr Trp Leu Glu Pro Leu Thr Lys Tyr Met Ph - #e Leu Thr Asp Asp Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Gly Leu Ile Met Met Ala Pro Ala Gln Ph - #e Asn Gln Gly Gln Tyr         #               175                                                           - Arg Arg Val Ile Thr Ile Asp Gly Ser Met Ph - #e Tyr Thr Asp Phe Met         #           190                                                               - Val Gln Leu Ser Pro Thr Pro Cys Trp Phe Al - #a Lys Pro Asp Arg Tyr         #       205                                                                   - Glu Glu Ile Leu His Glu Trp Cys Arg Asn Va - #l Lys Thr Ile Gly Leu         #   220                                                                       - Asp Gly Ala Arg Asp Tyr His Tyr Tyr Trp Va - #l Pro Tyr Asn Pro Gln         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro His His Lys Ala Val Leu Leu Tyr Trp Ty - #r Arg Thr His Gly Arg         #               255                                                           - Glu Pro Pro Val Arg Phe Gln Glu Ala Ile Ar - #g Tyr Asp Arg Pro Ala         #           270                                                               - Ile Pro Ser Gly Ser Glu Asp Ser Lys Arg Se - #r Asn Asp Ser Arg Gly         #       285                                                                   - Glu Ser Ser Gly Pro Asn Trp Ile Asp Ile Gl - #u Asn Tyr Thr Pro Lys         #   300                                                                       - Asn Asn Val Pro Ile Ile Ile Ser Asp Asp As - #p Val Pro Thr Ala Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Lys Gly Met Asn Asn Gln Ser Val Val Il - #e Pro Ala Ile Val Leu         #               335                                                           - Ser Cys Leu Ile Ile Ala Leu Ile Leu Gly Va - #l Ile Tyr Tyr Ile Leu         #           350                                                               - Arg Val Lys Arg Ser Arg Ser Thr Ala Tyr Gl - #n Gln Leu Pro Ile Ile         #       365                                                                   - His Thr Thr His His Pro                                                         370                                                                       - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 442 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Met Pro Ala Val Leu Leu Val Leu Tyr Val As - #n Pro Pro Pro Ser Val         #                15                                                           - Cys Ile Leu Thr Gln Lys Leu Ser Leu Gly Le - #u Tyr Asn Gln Trp Trp         #            30                                                               - Arg Val Cys Arg Ser Val Pro Pro Pro Trp Ty - #r Val Phe Phe Asn Lys         #        45                                                                   - Arg Ser Met Ser Thr Phe Lys Leu Met Met As - #p Gly Arg Leu Val Phe         #    60                                                                       - Ala Met Ala Ile Ala Ile Leu Ser Val Val Le - #u Ser Cys Gly Thr Cys         #80                                                                           - Glu Lys Ala Lys Arg Ala Val Arg Gly Arg Gl - #n Asp Arg Pro Lys Glu         #                95                                                           - Phe Pro Pro Pro Arg Tyr Asn Tyr Thr Ile Le - #u Thr Arg Tyr Asn Ala         #           110                                                               - Thr Ala Leu Ala Ser Pro Phe Ile Asn Asp Gl - #n Val Lys Asn Val Asp         #       125                                                                   - Leu Arg Ile Val Thr Ala Thr Arg Pro Cys Gl - #u Met Ile Ala Leu Ile         #   140                                                                       - Ala Lys Thr Asn Ile Asp Ser Ile Leu Lys Gl - #u Leu Ala Ala Ala Gln         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Thr Tyr Ser Ala Arg Leu Thr Trp Phe Ly - #s Ile Met Pro Thr Cys         #               175                                                           - Ala Thr Pro Ile His Asp Val Ser Tyr Met Ly - #s Cys Asn Pro Lys Leu         #           190                                                               - Ser Phe Ala Met Cys Asp Glu Arg Ser Asp Il - #e Leu Trp Gln Ala Ser         #       205                                                                   - Leu Ile Thr Met Ala Ala Glu Thr Asp Asp Gl - #u Leu Gly Leu Val Leu         #   220                                                                       - Ala Ala Pro Ala His Ser Ala Ser Gly Leu Ty - #r Arg Arg Val Ile Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Asp Gly Arg Arg Ile Tyr Thr Asp Phe Se - #r Val Thr Ile Pro Ser         #               255                                                           - Glu Arg Cys Pro Ile Ala Phe Glu Leu Asn Ph - #e Gly Asn Pro Asp Arg         #           270                                                               - Cys Lys Thr Pro Glu Gln Tyr Ser Arg Gly Gl - #u Val Phe Thr Arg Arg         #       285                                                                   - Phe Leu Gly Glu Phe Asn Phe Pro Gln Gly Gl - #u His Met Thr Trp Val         #   300                                                                       - Lys Phe Trp Phe Val Tyr Asp Gly Gly Asn Le - #u Pro Val Gln Phe Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Gln Ala Phe Ala Arg Pro Val Pro Pr - #o Asp Asn His Pro Gly         #               335                                                           - Phe Asp Ser Val Glu Ser Glu Ile Thr Gln As - #n Lys Thr Asp Pro Lys         #           350                                                               - Pro Gly Gln Ala Asp Pro Lys Pro Asn Gln Pr - #o Phe Lys Trp Pro Ser         #       365                                                                   - Ile Lys His Leu Val Pro Arg Leu Asp Glu Va - #l Asp Glu Val Ile Glu         #   380                                                                       - Pro Val Thr Lys Pro Pro Lys Thr Ser Lys Se - #r Asn Ser Thr Phe Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Ile Ser Val Gly Leu Gly Ile Ala Gly Le - #u Val Leu Val Gly Val         #               415                                                           - Ile Leu Tyr Val Cys Leu Arg Arg Lys Lys Gl - #u Leu Lys Val Cys Thr         #           430                                                               - Glu Arg Leu Asp Ser Pro Thr Leu Asp Leu                                     #       440                                                                   - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 393 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Va - #l Ile Leu Phe Val Val         #                15                                                           - Ile Val Gly Leu His Gly Val Arg Gly Lys Ty - #r Ala Leu Ala Asp Ala         #            30                                                               - Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Ar - #g Gly Lys Asp Leu Pro         #        45                                                                   - Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Va - #l Arg Arg Val Tyr His         #    60                                                                       - Ile Gln Ala Gly Leu Pro Asn Pro Phe Gln Pr - #o Pro Ser Leu Pro Ile         #80                                                                           - Thr Val Tyr Arg Arg Val Glu Arg Ala Cys Ar - #g Ser Val Leu Leu Asn         #                95                                                           - Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gl - #y Ala Ser Glu Asp Val         #           110                                                               - Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Tr - #p Phe Arg Met Gly Gly         #       125                                                                   - Asn Cys Ala Ile Pro Ile Thr Val Met Glu Ty - #r Thr Glu Cys Ser Tyr         #   140                                                                       - Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Th - #r Gln Pro Arg Trp Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu As - #p Asn Leu Gly Phe Leu         #               175                                                           - Met His Ala Pro Ala Phe Glu Thr Ala Gly Th - #r Tyr Leu Arg Leu Val         #           190                                                               - Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Ph - #e Ile Leu Glu His Arg         #       205                                                                   - Ala Lys Gly Ser Cys Lys Tyr Thr Leu Pro Le - #u Arg Ile Pro Pro Ser         #   220                                                                       - Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gl - #y Val Thr Val Asp Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Gly Met Leu Pro Arg Phe Ile Pro Glu As - #n Gln Arg Thr Val Ala         #               255                                                           - Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gl - #y Pro Arg Ala Pro Tyr         #           270                                                               - Thr Ser Thr Leu Leu Pro Pro Glu Leu Pro Gl - #u Thr Pro Asn Ala Thr         #       285                                                                   - Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu As - #p Ser Ala Leu Leu Glu         #   300                                                                       - Asp Pro Val Gly Thr Val Ala Pro Gln Ile Pr - #o Pro Asn Trp His Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Ser Ile Gln Asp Ala Ala Thr Pro Tyr Hi - #s Pro Pro Ala Thr Pro         #               335                                                           - Asn Asn Met Gly Leu Ile Ala Gly Ala Val Gl - #y Gly Ser Leu Leu Ala         #           350                                                               - Ala Leu Val Ile Cys Gly Ile Val Tyr Trp Me - #t Arg Arg Arg Thr Arg         #       365                                                                   - Lys Ala Pro Lys Arg Ile Arg Leu Pro His Il - #e Arg Glu Asp Asp Gln         #   380                                                                       - Pro Ser Ser His Gln Pro Leu Phe Tyr                                         385                 3 - #90                                                   - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                # 20               CGGG                                                       - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #             28   ATGG GCCCTTAA                                              - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 73 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - AGCTTCCCGG GTAAGTAATA CGTCAAGGAG AAAACGAAAC GATCTGTAGT TA - #GCGGCCGC         60                                                                          #      73                                                                     - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 69 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - AGGGCCCATT CATTATGCAG TTCCTCTTTT GCTTTGCTAG ACATCAATCG CC - #GGCGGATT         60                                                                          #         69                                                                  - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                # 20               CCGC                                                       - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #                 22CCG TT                                                    - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                # 20               GCAA                                                       - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #   41             GCTC CCCGGGCTCG AGGGATCCGT T                               - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #    39            AGGG GCCCGAGCTC CCTAGGCAA                                  - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 75 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - TATGAGTAAC TTAACTCTTT TGTTAATTAA AAGTATATTC AAAAAATAAG TT - #ATATAAAT         60                                                                          #    75                                                                       - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 73 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - ACTCATTGAA TTGAGAAAAC AATTAATTTT CATATAAGTT TTTTATTCAA TA - #TATTTATC         60                                                                          #      73                                                                     - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 49 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #               49TGTTA ACTTTATATA ACTTATTTTT TGAATATAC                       - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 67 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - ACACGAATGA TTTTCTAAAG TATTTGGAAA GTTTTATAGG TAGTTGATAG AA - #CAAAATAC         60                                                                          #          67                                                                 - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #             51AGATTTC ATAAACCTTT CAAAATATCC ATCAACTATC T                    - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #                 46TTT TATACTAAGA TCTCCCGGGC TGCAGC                          - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - TGTTTTATGT ATTAAAACAT TTTTATTTAG TGAAAAATAT GATTCTAGAG GG - #CCCGACGT         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #              50CACCAA TTTAGATCTT ACTCAAAATA TGTAACAATA                      - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                # 44               ACTC ATATTAATAA AAATAATATT TATT                            - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                - GATCCTGAGT ACTTTGTAAT ATAATGATAT ATATTTTCAC TTTATCTCAT TT - #GAGAATAA         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - GACTCATGAA ACATTATATT ACTATATATA AAAGTGAAAT AGAGTAAACT CT - #TATTTTTC         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                - GATCCAGATC TCCCGGGAAA AAAATTATTT AACTTTTCAT TAATAGGGAT TT - #GACGTATG         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                - GTCTAGAGGG CCCTTTTTTT AATAAATTGA AAAGTAATTA TCCCTAAACT GC - #ATACTACG         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #    40            TGCA GGGCGCCGGA TCCTTTTTCT                                 - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #    40            ACGT CCCGCGGCCT AGGAAAAAGA                                 - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 59 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                - CGATATCCGT TAAGTTTGTA TCGTAATGGG CTCCAGATCT TCTACCAGGA TC - #CCGGTAC          59                                                                          - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 55 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                - CGGGATCCTG GTAGAAGATC TGGAGCCCAT TACGATACAA ACTTAACGGA TA - #TCG              55                                                                          - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 13 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #              26  AACT ACGTAG                                                - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #        34        CTTT TTATAAAAAG AGCT                                       - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                # 20               ATCC                                                       - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                # 44               TATA AAAATCATAT TTTTGTAGTG GCTC                            - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 67 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                - AATTCAGGAT CGTTCCTTTA CTAGTTGAGA TTCTCAAGGA TGATGGGATT TA - #ATTTTTAT         60                                                                          #          67                                                                 - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 67 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                - AATTCAAGCT TATAAAAATT AAATCCCATC ATCCTTGAGA ATCTCAACTA GT - #AAAGGAAC         60                                                                          #          67                                                                 - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                - CTAGACACTT TATGTTTTTT AATATCCGGT CTTAAAAGCT TCCCGGGGAT CC - #TTATACGG         60                                                                          #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 65 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                - ATTATTCCCC GTATAAGGAT CCCCCGGGAA GCTTTTAAGA CCGGATATTA AA - #AAACATAA         60                                                                          #            65                                                               - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3209 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                - TGAATGTTAA ATGTTATACT TTGGATGAAG CTATAAATAT GCATTGGAAA AA - #TAATCCAT         60                                                                          - TTAAAGAAAG GATTCAAATA CTACAAAACC TAAGCGATAA TATGTTAACT AA - #GCTTATTC        120                                                                          - TTAACGACGC TTTAAATATA CACAAATAAA CATAATTTTT GTATAACCTA AC - #AAATAACT        180                                                                          - AAAACATAAA AATAATAAAA GGAAATGTAA TATCGTAATT ATTTTACTCA GG - #AATGGGGT        240                                                                          - TAAATATTTA TATCACGTGT ATATCTATAC TGTTATCGTA TACTCTTTAC AA - #TTACTATT        300                                                                          - ACGAATATGC AAGAGATAAT AAGATTACGT ATTTAAGAGA ATCTTGTCAT GA - #TAATTGGG        360                                                                          - TACGACATAG TGATAAATGC TATTTCGCAT CGTTACATAA AGTCAGTTGG AA - #AGATGGAT        420                                                                          - TTGACAGATG TAACTTAATA GGTGCAAAAA TGTTAAATAA CAGCATTCTA TC - #GGAAGATA        480                                                                          - GGATACCAGT TATATTATAC AAAAATCACT GGTTGGATAA AACAGATTCT GC - #AATATTCG        540                                                                          - TAAAAGATGA AGATTACTGC GAATTTGTAA ACTATGACAA TAAAAAGCCA TT - #TATCTCAA        600                                                                          - CGACATCGTG TAATTCTTCC ATGTTTTATG TATGTGTTTC AGATATTATG AG - #ATTACTAT        660                                                                          - AAACTTTTTG TATACTTATA TTCCGTAAAC TATATTAATC ATGAAGAAAA TG - #AAAAAGTA        720                                                                          - TAGAAGCTGT TCACGAGCGG TTGTTGAAAA CAACAAAATT ATACATTCAA GA - #TGGCTTAC        780                                                                          - ATATACGTCT GTGAGGCTAT CATGGATAAT GACAATGCAT CTCTAAATAG GT - #TTTTGGAC        840                                                                          - AATGGATTCG ACCCTAACAC GGAATATGGT ACTCTACAAT CTCCTCTTGA AA - #TGGCTGTA        900                                                                          - ATGTTCAAGA ATACCGAGGC TATAAAAATC TTGATGAGGT ATGGAGCTAA AC - #CTGTAGTT        960                                                                          - ACTGAATGCA CAACTTCTTG TCTGCATGAT GCGGTGTTGA GAGACGACTA CA - #AAATAGTG       1020                                                                          - AAAGATCTGT TGAAGAATAA CTATGTAAAC AATGTTCTTT ACAGCGGAGG CT - #TTACTCCT       1080                                                                          - TTGTGTTTGG CAGCTTACCT TAACAAAGTT AATTTGGTTA AACTTCTATT GG - #CTCATTCG       1140                                                                          - GCGGATGTAG ATATTTCAAA CACGGATCGG TTAACTCCTC TACATATAGC CG - #TATCAAAT       1200                                                                          - AAAAATTTAA CAATGGTTAA ACTTCTATTG AACAAAGGTG CTGATACTGA CT - #TGCTGGAT       1260                                                                          - AACATGGGAC GTACTCCTTT AATGATCGCT GTACAATCTG GAAATATTGA AA - #TATGTAGC       1320                                                                          - ACACTACTTA AAAAAAATAA AATGTCCAGA ACTGGGAAAA ATTGATCTTG CC - #AGCTGTAA       1380                                                                          - TTCATGGTAG AAAAGAAGTG CTCAGGCTAC TTTTCAACAA AGGAGCAGAT GT - #AAACTACA       1440                                                                          - TCTTTGAAAG AAATGGAAAA TCATATACTG TTTTGGAATT GATTAAAGAA AG - #TTACTCTG       1500                                                                          - AGACACAAAA GAGGTAGCTG AAGTGGTACT CTCAAAATGC AGAACGATGA CT - #GCGAAGCA       1560                                                                          - AGAAGTAGAG AAATAACACT TTATGACTTT CTTAGTTGTA GAAAAGATAG AG - #ATATAATG       1620                                                                          - ATGGTCATAA ATAACTCTGA TATTGCAAGT AAATGCAATA ATAAGTTAGA TT - #TATTTAAA       1680                                                                          - AGGATAGTTA AAAATAGAAA AAAAGAGTTA ATTTGTAGGG TTAAAATAAT AC - #ATAAGATC       1740                                                                          - TTAAAATTTA TAAATACGCA TAATAATAAA AATAGATTAT ACTTATTACC TT - #CAGAGATA       1800                                                                          - AAATTTAAGA TATTTACTTA TTTAACTTAT AAAGATCTAA AATGCATAAT TT - #CTAAATAA       1860                                                                          - TGAAAAAAAA GTACATCATG AGCAACGCGT TAGTATATTT TACAATGGAG AT - #TAACGCTC       1920                                                                          - TATACCGTTC TATGTTTATT GATTCAGATG ATGTTTTAGA AAAGAAAGTT AT - #TGAATATG       1980                                                                          - AAAACTTTAA TGAAGATGAA GATGACGACG ATGATTATTG TTGTAAATCT GT - #TTTAGATG       2040                                                                          - AAGAAGATGA CGCGCTAAAG TATACTATGG TTACAAAGTA TAAGTCTATA CT - #ACTAATGG       2100                                                                          - CGACTTGTGC AAGAAGGTAT AGTATAGTGA AAATGTTGTT AGATTATGAT TA - #TGAAAAAC       2160                                                                          - CAAATAAATC AGATCCATAT CTAAAGGTAT CTCCTTTGCA CATAATTTCA TC - #TATTCCTA       2220                                                                          - GTTTAGAATA CTTTTCATTA TATTTGTTTA CAGCTGAAGA CGAAAAAAAT AT - #ATCGATAA       2280                                                                          - TAGAAGATTA TGTTAACTCT GCTAATAAGA TGAAATTGAA TGAGTCTGTG AT - #AATAGCTA       2340                                                                          - TAATCAGAGA AGTTCTAAAA GGAAATAAAA ATCTAACTGA TCAGGATATA AA - #AACATTGG       2400                                                                          - CTGATGAAAT CAACAAGGAG GAACTGAATA TAGCTAAACT ATTGTTAGAT AG - #AGGGGCCA       2460                                                                          - AAGTAAATTA CAAGGATGTT TACGGTTCTT CAGCTCTCCA TAGAGCTGCT AT - #TGGTAGGA       2520                                                                          - AACAGGATAT GATAAAGCTG TTAATCGATC ATGGAGCTGA TGTAAACTCT TT - #AACTATTG       2580                                                                          - CTAAAGATAA TCTTATTAAA AAAAAATAAT ATCACGTTTA GTAATATTAA AA - #TATATTAA       2640                                                                          - TAACTCTATT ACTAATAACT CCAGTGGATA TGAACATAAT ACGAAGTTTA TA - #CATTCTCA       2700                                                                          - TCAAAATCTT ATTGACATCA AGTTAGATTG TGAAAATGAG ATTATGAAAT TA - #AGGAATAC       2760                                                                          - AAAAATAGGA TGTAAGAACT TACTAGAATG TTTTATCAAT AATGATATGA AT - #ACAGTATC       2820                                                                          - TAGGGCTATA AACAATGAAA CGATTAAAAA TTATAAAAAT CATTTCCCTA TA - #TATAATAC       2880                                                                          - GCTCATAGAA AAATTCATTT CTGAAAGTAT ACTAAGACAC GAATTATTGG AT - #GGAGTTAT       2940                                                                          - AAATTCTTTT CAAGGATTCA ATAATAAATT GCCTTACGAG ATTCAGTACA TT - #ATACTGGA       3000                                                                          - GAATCTTAAT AACCATGAAC TAAAAAAAAT TTTAGATAAT ATACATTAAA AA - #GGTAAATA       3060                                                                          - GATCATCTGT TATTATAAGC AAAGATGCTT GTTGCCAATA ATATACAACA GG - #TATTTGTT       3120                                                                          - TTTATTTTTA ACTACATATT TGATGTTCAT TCTCTTTATA TAGTATACAC AG - #AAAATTCA       3180                                                                          #          3209    TCTA GTTATCTAG                                             - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #            29    AGCT AGCTAGTTT                                             - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                #                 46GGG AATTCTAGCT AGCTAGTTTT TATAAA                          - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #              50CTAGCT AGCTAGAATT CCCGGGAAGC TTTTGAGAGT                      - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 71 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                - CTGAAATTAT TTCATTATCG CGATATCCGT TAAGTTTGTA TCGTAATGGT TC - #CTCAGGCT         60                                                                          #       71                                                                    - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                #                48TAAC GGATATCGCG ATAATGAAAT AATTTCAG                        - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 73 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                - ACCCCTTCTG GTTTTTCCGT TGTGTTTTGG GAAATTCCCT ATTTACACGA TC - #CCAGACAA         60                                                                          #      73                                                                     - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                #             51TTGTCTG GGATCGTGTA AATAGGGAAT TTCCCAAAAC A                    - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                #45                AGGG GTACAAACAG GAGAGCCTGA GGAAC                           - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 11 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3659 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                - GATATCTGTG GTCTATATAT ACTACACCCT ACCGATATTA ACCAACGAGT TT - #CTCACAAG         60                                                                          - AAAACTTGTT TAGTAGATAG AGATTCTTTG ATTGTGTTTA AAAGAAGTAC CA - #GTAAAAAG        120                                                                          - TGTGGCATAT GCATAGAAGA AATAAACAAA AAACATATTT CCGAACAGTA TT - #TTGGAATT        180                                                                          - CTCCCAAGTT GTAAACATAT TTTTTGCCTA TCATGTATAA GACGTTGGGC AG - #ATACTACC        240                                                                          - AGAAATACAG ATACTGAAAA TACGTGTCCT GAATGTAGAA TAGTTTTTCC TT - #TCATAATA        300                                                                          - CCCAGTAGGT ATTGGATAGA TAATAAATAT GATAAAAAAA TATTATATAA TA - #GATATAAG        360                                                                          - AAAATGATTT TTACAAAAAT ACCTATAAGA ACAATAAAAA TATAATTACA TT - #TACGGAAA        420                                                                          - ATAGCTGGTT TTAGTTTACC AACTTAGAGT AATTATCATA TTGAATCTAT AT - #TGTTTTTT        480                                                                          - AGTTATATAA AAACATGATT AGCCCCCAAT CGGATGAAAA TATAAAAGAT GT - #TGAGAATT        540                                                                          - TCGAATACAA CAAAAAGAGG AATCGTACGT TGTCCATATC CAAACATATA AA - #TAAAAATT        600                                                                          - CAAAAGTAGT ATTATACTGG ATGTTTAGAG ATCAACGTGT ACAAGATAAT TG - #GGCTTTAA        660                                                                          - TTTACGCACA ACGATTAGCG TTAAAACTCA AAATACCTCT AAGAATATGC TT - #TTGTGTCG        720                                                                          - TGCCAAAATT TCACACTACT ACTTCTAGAC ACTTTATGTT TTTAATATCC GG - #TCTTAAAG        780                                                                          - AAGTCGCGGA AGAATGTAAA AGACTATGTA TAGGGTTTTC ATTGATATAT GG - #CGTACCAA        840                                                                          - AAGTAATAAT TCCGTGTATA GTAAAAAAAT ACAGAGTCGG AGTAATCATA AC - #GGATTTCT        900                                                                          - TTCCATTACG TGTTCCCGAA AGATTAATGA AACAGACTGT AATATCTCTT CC - #AGATAACA        960                                                                          - TACCTTTTAT ACAAGTAGAC GCTCATAATA TAGTACCTTG TTGGGAAGCT TC - #TGATAAAG       1020                                                                          - AAGAATACGG TGCACGAACT TTAAGAAAAA AGATATTTGA TAAATTATAT GA - #ATATATGA       1080                                                                          - CAGAATTTCC TGTTGTTCGT AAACATCCAT ACGGTCCATT TTCTATATCT AT - #TGCAAAAC       1140                                                                          - CCAAAAATAT ATCATTAGAC AAGACGGTAT TACCCGTAAA ATGGGCAACG CC - #TGGAACAA       1200                                                                          - AAGCTGGAAT AATTGTTTTA AAAGAATTTA TAAAAAACAG ATTACCGTCA TA - #CGACGCGG       1260                                                                          - ATCATAACAA TCCTACGTGT GACGCTTTGA GTAACTTATC TCCGTGGCTA CA - #TTTTGGTC       1320                                                                          - ATGTATCCGC ACAACGTGTT GCCTTAGAAG TATTAAAATG TATACGAGAA AG - #CAAAAAAA       1380                                                                          - ACGTTGAAAC GTTTATAGAT GAAATAATTG TAAGAAGAGA ACTATCGGAT AA - #TTTTTGTT       1440                                                                          - ACTATAACAA ACATTATGAT AGTATCCAGT CTACTCATTC ATGGGTTAGA AA - #AACATTAG       1500                                                                          - AAGATCACAT TAATGATCCT AGAAAGTATA TATATTCCAT TAAACAACTC GA - #AAAAGCGG       1560                                                                          - AAACTCATGA TCCTCTATGG AACGCGTCAC AAATGCAGAT GGTGAGAGAA GG - #AAAAATGC       1620                                                                          - ATAGTTTTTT ACGAATGTAT TGGGCTAAGA AGATACTTGA ATGGACTAGA AC - #ACCTGAAG       1680                                                                          - ACGCTTTGAG TTATAGTATC TATTTGAACA ACAAGTACGA ACTAGACGGC AC - #GGATCCTA       1740                                                                          - ACGGATACGT AGGTTGTATG TGGTCTATTT GCGGATTACA CGATAGAGCG TG - #GAAAGCAA       1800                                                                          - GACCGATATT TGGAAAGATA AGATATATGA ATTATGAGAG TTCTAAGAAG AA - #ATTTGATG       1860                                                                          - TTGCTGTATT TATACAGAAA TACAATTAAG ATAAATAATA TACAGCATTG TA - #ACCATCGT       1920                                                                          - CATCCGTTAT ACGGGGAATA ATATTACCAT ACAGTATTAT TAAATTTTCT TA - #CGAAGAAT       1980                                                                          - ATAGATCGGT ATTTATCGTT AGTTTATTTT ACATTTATTA ATTAAACATG TC - #TACTATTA       2040                                                                          - CCTGTTATGG AAATGACAAA TTTAGTTATA TAATTTATGA TAAAATTAAG AT - #AATAATAA       2100                                                                          - TGAAATCAAA TAATTATGTA AATGCTACTA GATTATGTGA ATTACGAGGA AG - #AAAGTTTA       2160                                                                          - CGAACTGGAA AAAATTAAGT GAATCTAAAA TATTAGTCGA TAATGTAAAA AA - #AATAAATG       2220                                                                          - ATAAAACTAA CCAGTTAAAA ACGGATATGA TTATATACGT TAAGGATATT GA - #TCATAAAG       2280                                                                          - GAAGAGATAC TTGCGGTTAC TATGTACACC AAGATCTGGT ATCTTCTATA TC - #AAATTGGA       2340                                                                          - TATCTCCGTT ATTCGCCGTT AAGGTAAATA AAATTATTAA CTATTATATA TG - #TAATGAAT       2400                                                                          - ATGATATACG ACTTAGCGAA ATGGAATCTG ATATGACAGA AGTAATAGAT GT - #AGTTGATA       2460                                                                          - AATTAGTAGG AGGATACAAT GATGAAATAG CAGAAATAAT ATATTTGTTT AA - #TAAATTTA       2520                                                                          - TAGAAAAATA TATTGCTAAC ATATCGTTAT CAACTGAATT ATCTAGTATA TT - #AAATAATT       2580                                                                          - TTATAAATTT TATAAATTTT AATAAAAAAT ACAATAACGA CATAAAGATA TT - #TAATCTTT       2640                                                                          - AATTCTTGAT CTGAAAAACA CATCTATAAA ACTAGATAAA AAGTTATTCG AT - #AAAGATAA       2700                                                                          - TAATGAATCG AACGATGAAA AATTGGAAAC AGAAGTTGAT AAGCTAATTT TT - #TTCATCTA       2760                                                                          - AATAGTATTA TTTTATTGAA GTACGAAGTT TTACGTTAGA TAAATAATAA AG - #GTCGATTT       2820                                                                          - TTACTTTGTT AAATATCAAA TATGTCATTA TCTGATAAAG ATACAAAAAC AC - #ACGGTGAT       2880                                                                          - TATCAACCAT CTAACGAACA GATATTACAA AAAATACGTC GGACTATGGA AA - #ACGAAGCT       2940                                                                          - GATAGCCTCA ATAGAAGAAG CATTAAAGAA ATTGTTGTAG ATGTTATGAA GA - #ATTGGGAT       3000                                                                          - CATCCTCAAC GAAGAAATAG ATAAAGTTCT AAACTGGAAA AATGATACAT TA - #AACGATTT       3060                                                                          - AGATCATCTA AATACAGATG ATAATATTAA GGAAATCATA CAATGTCTGA TT - #AGAGAATT       3120                                                                          - TGCGTTTAAA AAGATCAATT CTATTATGTA TAGTTATGCT ATGGTAAAAC TC - #AATTCAGA       3180                                                                          - TAACGAACAT TGAAAGATAA AATTAAGGAT TATTTTATAG AAACTATTCT TA - #AAGACAAA       3240                                                                          - CGTGGTTATA AACAAAAGCC ATTACCCGGA TTGGAAACTA AAATACTAGA TA - #GTATTATA       3300                                                                          - AGATTTTAAA AACATAAAAT TAATAGGTTT TTATAGATTG ACTTATTATA TA - #CAATATGG       3360                                                                          - ATAAAAGATA TATATCAACT AGAAAGTTGA ATGACGGATT CTTAATTTTA TA - #TTATGATT       3420                                                                          - CAATAGAAAT TATTGTCATG TCGTGTAATC ATTTTATAAA TATATCAGCG TT - #ACTAGCTA       3480                                                                          - AGAAAAACAA GGACTTTAAT GAATGGCTAA AGATAGAATC ATTTAGAGAA AT - #AATAGATA       3540                                                                          - CTTTAGATAA AATTAATTAC GATCTAGGAC AACGATATTG TGAAGAACTT AC - #GGCGCATC       3600                                                                          - ACATTCCAGT GTAATTATTG AGGTCAAAGC TAGTAACTTA ATAGATGACA GG - #ACAGCTG        3659                                                                          - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                - TCATTATCGC GATATCCGTG TTAACTAGCT AGCTAATTTT TATTCCCGGG AT - #CCTTATCA         60                                                                          - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                - GTATAAGGAT CCCGGGAATA AAAATTAGCT AGCTAGTTAA CACGGATATC GC - #GATAATGA         60                                                                          - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2356 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                - TGTCTGGACT AACTGATTTC ATGGAACAAT TTTCATCAAA AATATCAGTT AT - #ACCTAGTT         60                                                                          - CTACAAAGAC AGAACTTTGA TGTTATGTTT GTGTTTGTAT AGAAAATTTT GG - #GATACTAA        120                                                                          - CTGATATTTC TGAATATTTC TGAATATTTC ATGTTACTTA CTTACTCCTA TC - #TTAGACGA        180                                                                          - TAATAAAATT CGAGGCGTAA TATGTTTTTC CAAATATTTG AAATTCTTAT AC - #GTATCGGC        240                                                                          - GAAGAAAAGT AACATACTAT AAGTGTTATG CAAGTAAGGT ATGTTAATGA TA - #TTGGATTT        300                                                                          - AATTTCATTG ACAATACATA TGTCCAAACA TTCCACTCGT AATTATGTAC GG - #AACGACTT        360                                                                          - TAGTTAAATA CTTAGTCACA AAAAACTTAT GACTGTCATT ATCTGAAAAC GG - #TGATTCCC        420                                                                          - ATAAATCAGA ATACTTAATA TTAAATAGAA TGCTCGCTTC TGGAGGTTTC CG - #GATACTAG        480                                                                          - ATAACATATC TTCTGTATTA TAGTTTAATT CACTCATTTT ATTACATAAT AC - #AGTAACAT        540                                                                          - CTCCCGAAAC CAATGATGTT ATATTAGATT TACTTACATA CTTCTTGTAA CT - #ATCATGAA        600                                                                          - TACGTTTGTT ATGATCTATA AAGAAGATGG ATGTATATTC TGTTCTAGAT AG - #CAAGTTCT        660                                                                          - TTAAGTTATT CTTTGTCTGT ATTACTATCA TCGTCTTCAT CATCGTCTAA AG - #GTAGCATT        720                                                                          - ATATAATAAA TCTAATAGTT GATTTCTCGA TCTATCAGTA CTCGCTTTCA AT - #AACATTTT        780                                                                          - TACTATAAGC ATAATAGAAG GCGGTGATAT CACTATATTT TTATCGGGTA TT - #CTTTTAGT        840                                                                          - AATTAGTTAG TTCGTAGAAT TTCGTAGAGA TAAAAGCCAA TTTGTTGTTG AT - #ACTGCTTA        900                                                                          - CGTTACTCAT GTTTCTTGTT TCTGTTAATT AACAGGTATA CCCTTACAAT AA - #GTTTAATT        960                                                                          - AACTTTTAGG TTTTTGTGAA GAACTTTTAG CTTCTAGTTC CCTTATCCAT AA - #TTGGGTCT       1020                                                                          - TAGATCTAGA TTCTTCCCAT GTATAAAGGG GGACATACCC AAAATCTTTA AA - #TGCTTTGT       1080                                                                          - CCGTTTCTAT AGTAAATGTC GTACATTCCT TAATCAAAGT ATAAGGATTT AG - #TAAAGGCG       1140                                                                          - TGTAAGAACA AATAGGTGAT AGTAATACTC TTAAACCTTT ATTAATATTA GC - #GATAAACC       1200                                                                          - TTAAACACCA TAAAGGAAGA CATGTATTCC GTAGATCCAT CCCTAATTGA TT - #AAAGAAAT       1260                                                                          - GCATGTTAAA ATCATGATAA TGTTCAGTAG GAGAGGTATC GTAACAGTAA TA - #CACGTTAT       1320                                                                          - TGCAGAGAGG ACTATGTTGA CCATTTTCTA TCATATTTCT TGCTGCTAAA AT - #ATGCATCC       1380                                                                          - AAGCTACGTT TCCTGCATAG ACTCTGCTAT GAAATACTTT ATCATCCGCA TA - #TTTATACA       1440                                                                          - TTTTCCTGCT TTTATACGAT CTTCTGTATA AAGTTTCTAG TACTGGACAG TA - #TTCTCCGA       1500                                                                          - AAACACCTAA TGGGCGTAGC GACAAGTGCA TAATCTAAGT CCTATATTAG AC - #ATAGTACC       1560                                                                          - GTTAGCTTCT AGTATATATT TCTCAGATAA CTTGTTTACT AAGAGGATAA GC - #CTCTTTAT       1620                                                                          - GGTTAGATTG ATAATACGTA TTCTCGTTTC CTCTTATCAT CGCATCTCCG GA - #GAAAGTTA       1680                                                                          - GGACCTACCG CAGAATAACT ACTCGTATAT ACTAAGACTC TTACGCCGTT AT - #ACAGACAA       1740                                                                          - GAATCTACTA CGTTCTTCGT TCCGTTGATA TTAACGTCCA TTATAGAGTC GT - #TAGTAAAC       1800                                                                          - TTACCCGCTA CATCATTTAT CGAAGCAATA TGAATGACCA CATCTGCTGA TC - #TAAGCGCT       1860                                                                          - TCGTCCAAAG TACTTTTATT TCTAACATCT CCAATCACGG GAACTATCTT TA - #TTATATTA       1920                                                                          - CATTTTTCTA CAAGATCTAG TAACCATTGG TCGATTCTAA TATCGTAAAC AC - #GAACTTCT       1980                                                                          - TTTTAAAGAG GATTCGAACA AGATAAGATT ATTTATAATG TGTCTACCTA AA - #AATCCACA       2040                                                                          - CCCTCCGGTT ACCACGTATA CTAGTGTACG CATTTTGAGT ATTAACTATA TA - #AGACCAAA       2100                                                                          - ATTATATTTT CATTTTCTGT TATATTATAC TATATAATAA AAACAAATAA AT - #ATACGAAT       2160                                                                          - ATTATAAGAA ATTTAGAACA CGTTATTAAA GTATTGCCTT TTTTATTAAC GG - #CGTGTTCT       2220                                                                          - TGTAATTGCC GTTTAGAATA GTCTTTATTT ACTTTAGATA ACTCTTCTAT CA - #TAACCGTC       2280                                                                          - TCCTTATTCC AATCTTCTTC AGAAGTACAT GAGTACTTAC CGAAGTTTAT CA - #TCATAGAG       2340                                                                          #  2356                                                                       - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                #                24TATT AGAC                                                  - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                #  18              AC                                                         - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                #  18              CG                                                         - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                #            29    AAGG GTATACCTG                                             - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 61 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                - AACGATTAGT TAGTTACTAA AAGCTTGCTG CAGCCCGGGT TTTTTATTAG TT - #TAGTTAGT         60                                                                          #               61                                                            - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                - GACTAACTAA CTAATAAAAA ACCCGGGCTG CAGCAAGCTT TTTGTAACTA AC - #TAATCGTT         60                                                                          - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 99 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                - GCACGGAACA AAGCTTATCG CGATATCCGT TAAGTTTGTA TCGTAATGCT AT - #CAATCACG         60                                                                          #    99            TAGC AGAGGGCTCA TCTCAGAAT                                  - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 99 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                - ATTCTGAGAT GAGCCCTCTG CTATGAGCAG GAACAGAATC GTGATTGATA GC - #ATTACGAT         60                                                                          #    99            TCGC GATAAGCTTT GTTCCGTGC                                  - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                - GAAAAATTTA AAGTCGACCT GTTTTGTTGA GTTGTTTGCG TGGTAACCAA TG - #CAAATCTG         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                - TCTAGCAAGA CTGACTATTG CAAAAAGAAG CACTATTTCC TCCATTACGA TA - #CAAACTTA         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 87 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                - ATCCGTTAAG TTTGTATCGT AATGGAGGAA ATAGTGCTTC TTTTTGCAAT AG - #TCAGTCTT         60                                                                          #             87   TTTG CATTGGT                                               - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 49 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                #               49TCAAC AAAACAGGTC GACTTTAAAT TTTTCTGCA                       - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 132 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                - GTACAGGTCG ACAAGCTTCC CGGGTATCGC GATATCCGTT AAGTTTGTAT CG - #TAATGAAT         60                                                                          - ACTCAAATTC TAATACTCAC TCTTGTGGCA GCCATTCACA CAAATGCAGA CA - #AAATCTGC        120                                                                          #      132                                                                    - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 132 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                - ATGATGTCCA AGGCAGATTT TGTCTGCATT TGTGTGAATG GCTGCCACAA GA - #GTGAGTAT         60                                                                          - TAGAATTTGA GTATTCATTA CGATACAAAC TTAACGGATA TCGCGATACC CG - #GGAAGCTT        120                                                                          #      132                                                                    - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                #             51CACCATT TGTATATAAG TTAACGAATT CCAAGTCAAG C                    - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                #             51TTCGTTA ACTTATATAC AAATGGTGCA CCGCATGTTA T                    - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                #            29    TACT TTCATTTTC                                             - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 88 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                - TTGTCGACTG AGATAAAGTG AAAATATATA TCATTATATT ACAAAGTACA AT - #TATTTAGG         60                                                                          #             88   TTGT ATCTATAT                                              - (2) INFORMATION FOR SEQ ID NO:95:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 56 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                - TTAGTACTTT CCGGTGTTGT TGGATCACAT ATTATTAAAG TATAAATAAT AA - #AGAA             56                                                                          - (2) INFORMATION FOR SEQ ID NO:96:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                # 20               AACT                                                       - (2) INFORMATION FOR SEQ ID NO:97:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                #21                ATTT C                                                     - (2) INFORMATION FOR SEQ ID NO:98:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                #             28   AACA CTAGAACC                                              - (2) INFORMATION FOR SEQ ID NO:99:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                #             27   CAAA ATTCCAT                                               - (2) INFORMATION FOR SEQ ID NO:100:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                               # 20               CACC                                                       - (2) INFORMATION FOR SEQ ID NO:101:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                               # 20               GCAC                                                       - (2) INFORMATION FOR SEQ ID NO:102:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 49 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                               #               49TCATA TGTCCCCTTT CTTTGGATTA CGAGATGGT                       - (2) INFORMATION FOR SEQ ID NO:103:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                               #       35         AGAA AGGGGACATA TGAAT                                      - (2) INFORMATION FOR SEQ ID NO:104:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2951 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                               - AGTACAATAA AAAGTATTAA ATAAAAATAC TTACTTACGA AAAAATGACT AA - #TTAGCTAT         60                                                                          - AAAAACCCGG GAAAGGATCC TGATCCTTTT TCTGGGTAAG TAATACGTCA AG - #GAGAAAAC        120                                                                          - GAAACGATCT GTAGTTAGCG GCCAAACTCG AGGTCGACTG AGATAAAGTG AA - #AATATATA        180                                                                          - TCATTATATT ACAAAGTACA ATTATTTAGG TTTAATCATG TTTTCATTGT AT - #CTATATAT        240                                                                          - TTTTTTTATT ATTTATACTT TAATAATATG TGATCCAACA ACACCGGAAA GT - #ACTATTAA        300                                                                          - TCCATTAAAT CATCACAATT TATCAACACC TAAACCTACT TCGGATGATA TT - #CGTGAAAT        360                                                                          - TTTACGTGAA TCCCAAATTG AATCTGATGA TACATCAACA TTTTACATGT GC - #CCACCACC        420                                                                          - ATCGGGATCA ACATTGGTGC GTTTGGAGCC ACCTAGAGCA TGTCCTAACT AT - #AAACTTGG        480                                                                          - TAAAAATTTT ACAGAAGGAA TTGCTGTAAT ATTTAAGGAA AATATTTCTC CT - #TATAAATT        540                                                                          - TAAAGCTAAT ATATACTACA AAAATATTAT TATCACCACT GTATGGTCTG GA - #AGCACATA        600                                                                          - TGCAGTAATT ACTAATAGAT ATACAGATCG TGTACCTATA GGTGTTCCTG AA - #ATTACAGA        660                                                                          - GTTGATTGAT AGAAGAGGTA TGTGTTTATC AAAAGCTGAT TATATTCGTA AT - #AATTATGA        720                                                                          - ATTTACCGCA TTTGATAAGG ATGAAGACCC CAGAGAAGTT CATTTAAAGC CT - #TCAAAGTT        780                                                                          - TAATACACCA GGATCCCGTG GATGGCATAC AGTTAATGAT ACTTACACAA AA - #ATTGGGGG        840                                                                          - TTCTGGATTT TATCATTCTG GAACATCTGT AAATTGTATA GTTGAAGAAG TT - #GATGCCAG        900                                                                          - ATCTGTTTAT CCATATGATT CATTTGCTAT CTCCACCGGG GATATAATTC AT - #ATGTCCCC        960                                                                          - TTTTTTTGGA TTACGAGATG GTGCTCATAC TGAATATATT AGTTATTCAA CT - #GATAGATT       1020                                                                          - TCAACAAATA GAAGGTTATT ATCCTATCGA CTTAGATACT AGACTACAGC TT - #GGTGCACC       1080                                                                          - AGTTTCTAGG AATTTTTTAA CAACACAACA CGTTACTGTT GCTTGGAATT GG - #GTTCCAAA       1140                                                                          - AATTCGTGAA GTGTGTACTT TGGCTAAATG GCGTGAAATT GATGAAATTA TT - #CGTGATGA       1200                                                                          - GTATAAGGGA TCTTACAGAT TTACAGCAAA ATCAATATCT GCAACATTTA TT - #TCTGATAC       1260                                                                          - TACTCAATTT GATATTGATC GTGTAAAGTT AAGTGATTGT GCCAAACGTG AA - #GCCATAGA       1320                                                                          - AGCTATTGAT AAGATCTACA AAAAAAAATA TAATAAAACT CATATTCAAA CA - #GGAGAATT       1380                                                                          - GGAAACATAC TTGGCTAGAG GGGGATTTAT TATAGCATTT AGACCAATGA TT - #AGTAATGA       1440                                                                          - GTTAGCAAAA TTGTATATAA ATGAGTTAGT AAGATCTAAT CGTACGGTTG AT - #TTGAAATC       1500                                                                          - TCTTTTAAAT CCATCTGTAA GAGGGGGGGC TAGAAAGAGA AGATCAGTAG AG - #GAAAATAA       1560                                                                          - AAGATCAAAA CGTAATATTG AAGGTGGTAT TGAAAATGTA AATAATTCAA CA - #ATAATTAA       1620                                                                          - GACAACTTCA TCTGTTCATT TTGCTATGCT TCAGTTTGCC TATGATCATA TT - #CAATCACA       1680                                                                          - TGTTAATGAA ATGCTTAGTA GAATTGCAAC TGCATGGTGT AATCTTCAAA AT - #AAAGAGAG       1740                                                                          - AACCCTTTGG AATGAAGTTA TGAAACTTAA TCCAACTAGT GTGGCTTCGG TT - #GCTATGGA       1800                                                                          - TCAAAGAGTT TCAGCACGAA TGTTAGGGGA TGTTCTTGCA GTTACTCAAT GT - #GTTAATAT       1860                                                                          - ATCAGGTTCT AGTGTTTTTA TTCAAAATTC CATGCGTGTT TTAGGGTCAA CA - #ACTACATG       1920                                                                          - TTACAGTCGT CCTCTTATAT CATTTAAAGC ACTAGAAAAC TCAACTAACT AT - #ATTGAAGG       1980                                                                          - ACAACTTGGG GAAAATAATG AACTATTAGT AGAACGAAAG CTAATTGAAC CA - #TGTACAGC       2040                                                                          - TAACCATAAA AGATATTTTA AATTTGGTGC AGATTATGTA TATTTTGAAA AC - #TATGCATA       2100                                                                          - TGTTCGAAAG GTACCTCTTA ATGAAATTGA AATGATCAGT GCATATGTAG AT - #CTTAATAT       2160                                                                          - TACATTACTT GAGGATCGTG AATTTTTACC ACTAGAGGTA TATACTCGAG CA - #GAGTTAGA       2220                                                                          - AGATACAGGA CTATTGGACT ATAGTGAGAT TCAACGTAGA AATCAACTAC AT - #GCACTTAA       2280                                                                          - GTTTTATGAT ATTGACAGTG TTGTAAAAGT TGATAATAAT GTTGTAATTA TG - #AGGGGCAT       2340                                                                          - TGCAAATTTT TTCCAAGGAC TTGGAGATGT TGGAGCGGGA TTTGGAAAAG TT - #GTTTTGGG       2400                                                                          - TGCTGCAAAT GCTGTTATTG CAACTGTTTC TGGAGTGTCC TCGTTTCTTA AT - #AACCCATT       2460                                                                          - TGGGGCGCTA GCCGTTGGAT TGCTGATTTT AGCTGGACTA TTTGCAGCGT TT - #TTGGCTTA       2520                                                                          - TAGATATGTT TCTAAACTTA AGTCAAATCC AATGAAAGCA CTATACCCAG TA - #ACTACAAA       2580                                                                          - AAATTTAAAA GAAAGTGTTA AGAATGGTAA TTCTGGAAAT AATAGTGATG GA - #GAAGAAAA       2640                                                                          - TGATGATAAT ATCGATGAAG AAAAGCTTCA ACAAGCTAAA GAAATGATTA AA - #TATATGTC       2700                                                                          - TCTAGTTTCT GCTATGGAAC AGCAGGAACA TAAAGCTATT AAAAAAAATA GT - #GGCCCTGC       2760                                                                          - CCTTCTAGCA AGTCACATTA CAAACCTATC TCTTAAACAT CGTGGTCCAA AA - #TACAAACG       2820                                                                          - TTTGAAAAAT GTAAATGAAA ATGAAAGTAA AGTTTAACCC GGGTACCGAG CT - #CGAATTCT       2880                                                                          - TTTTATTGAT TAACTAGTCA AATGAGTATA TATAATTGAA AAAGTAAAAT AT - #AAATCATA       2940                                                                          #     2951                                                                    - (2) INFORMATION FOR SEQ ID NO:105:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2951 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                               - TCATGTTATT TTTCATAATT TATTTTTATG AATGAATGCT TTTTTACTGA TT - #AATCGATA         60                                                                          - TTTTTGGGCC CTTTCCTAGG ACTAGGAAAA AGACCCATTC ATTATGCAGT TC - #CTCTTTTG        120                                                                          - CTTTGCTAGA CATCAATCGC CGGTTTGAGC TCCAGCTGAC TCTATTTCAC TT - #TTATATAT        180                                                                          - AGTAATATAA TGTTTCATGT TAATAAATCC AAATTAGTAC AAAAGTAACA TA - #GATATATA        240                                                                          - AAAAAAATAA TAAATATGAA ATTATTATAC ACTAGGTTGT TGTGGCCTTT CA - #TGATAATT        300                                                                          - AGGTAATTTA GTAGTGTTAA ATAGTTGTGG ATTTGGATGA AGCCTACTAT AA - #GCACTTTA        360                                                                          - AAATGCACTT AGGGTTTAAC TTAGACTACT ATGTAGTTGT AAAATGTACA CG - #GGTGGTGG        420                                                                          - TAGCCCTAGT TGTAACCACG CAAACCTCGG TGGATCTCGT ACAGGATTGA TA - #TTTGAACC        480                                                                          - ATTTTTAAAA TGTCTTCCTT AACGACATTA TAAATTCCTT TTATAAAGAG GA - #ATATTTAA        540                                                                          - ATTTCGATTA TATATGATGT TTTTATAATA ATAGTGGTGA CATACCAGAC CT - #TCGTGTAT        600                                                                          - ACGTCATTAA TGATTATCTA TATGTCTAGC ACATGGATAT CCACAAGGAC TT - #TAATGTCT        660                                                                          - CAACTAACTA TCTTCTCCAT ACACAAATAG TTTTCGACTA ATATAAGCAT TA - #TTAATACT        720                                                                          - TAAATGGCGT AAACTATTCC TACTTCTGGG GTCTCTTCAA GTAAATTTCG GA - #AGTTTCAA        780                                                                          - ATTATGTGGT CCTAGGGCAC CTACCGTATG TCAATTACTA TGAATGTGTT TT - #TAACCCCC        840                                                                          - AAGACCTAAA ATAGTAAGAC CTTGTAGACA TTTAACATAT CAACTTCTTC AA - #CTACGGTC        900                                                                          - TAGACAAATA GGTATACTAA GTAAACGATA GAGGTGGCCC CTATATTAAG TA - #TACAGGGG        960                                                                          - AAAAAAACCT AATGCTCTAC CACGAGTATG ACTTATATAA TCAATAAGTT GA - #CTATCTAA       1020                                                                          - AGTTGTTTAT CTTCCAATAA TAGGATAGCT GAATCTATGA TCTGATGTCG AA - #CCACGTGG       1080                                                                          - TCAAAGATCC TTAAAAAATT GTTGTGTTGT GCAATGACAA CGAACCTTAA CC - #CAAGGTTT       1140                                                                          - TTAAGCACTT CACACATGAA ACCGATTTAC CGCACTTTAA CTACTTTAAT AA - #GCACTACT       1200                                                                          - CATATTCCCT AGAATGTCTA AATGTCGTTT TAGTTATAGA CGTTGTAAAT AA - #AGACTATG       1260                                                                          - ATGAGTTAAA CTATAACTAG CACATTTCAA TTCACTAACA CGGTTTGCAC TT - #CGGTATCT       1320                                                                          - TCGATAACTA TTCTAGATGT TTTTTTTTAT ATTATTTTGA GTATAAGTTT GT - #CCTCTTAA       1380                                                                          - CCTTTGTATG AACCGATCTC CCCCTAAATA ATATCGTAAA TCTGGTTACT AA - #TCATTACT       1440                                                                          - CAATCGTTTT AACATATATT TACTCAATCA TTCTAGATTA GCATGCCAAC TA - #AACTTTAG       1500                                                                          - AGAAAATTTA GGTAGACATT CTCCCCCCCG ATCTTTCTCT TCTAGTCATC TC - #CTTTTATT       1560                                                                          - TTCTAGTTTT GCATTATAAC TTCCACCATA ACTTTTACAT TTATTAAGTT GT - #TATTAATT       1620                                                                          - CTGTTGAAGT AGACAAGTAA AACGATACGA AGTCAAACGG ATACTAGTAT AA - #GTTAGTGT       1680                                                                          - ACAATTACTT TACGAATCAT CTTAACGTTG ACGTACCACA TTAGAAGTTT TA - #TTTCTCTC       1740                                                                          - TTGGGAAACC TTACTTCAAT ACTTTGAATT AGGTTGATCA CACCGAAGCC AA - #CGATACCT       1800                                                                          - AGTTTCTCAA AGTCGTGCTT ACAATCCCCT ACAAGAACGT CAATGAGTTA CA - #CAATTATA       1860                                                                          - TAGTCCAAGA TCACAAAAAT AAGTTTTAAG GTACGCACAA AATCCCAGTT GT - #TGATGTAC       1920                                                                          - AATGTCAGCA GGAGAATATA GTAAATTTCG TGATCTTTTG AGTTGATTGA TA - #TAACTTCC       1980                                                                          - TGTTGAACCC CTTTTATTAC TTGATAATCA TCTTGCTTTC GATTAACTTG GT - #ACATGTCG       2040                                                                          - ATTGGTATTT TCTATAAAAT TTAAACCACG TCTAATACAT ATAAAACTTT TG - #ATACGTAT       2100                                                                          - ACAAGCTTTC CATGGAGAAT TACTTTAACT TTACTAGTCA CGTATACATC TA - #GAATTATA       2160                                                                          - ATGTAATGAA CTCCTAGCAC TTAAAAATGG TGATCTCCAT ATATGAGCTC GT - #CTCAATCT       2220                                                                          - TCTATGTCCT GATAACCTGA TATCACTCTA AGTTGCATCT TTAGTTGATG TA - #CGTGAATT       2280                                                                          - CAAAATACTA TAACTGTCAC AACATTTTCA ACTATTATTA CAACATTAAT AC - #TCCCCGTA       2340                                                                          - ACGTTTAAAA AAGGTTCCTG AACCTCTACA ACCTCGCCCT AAACCTTTTC AA - #CAAAACCC       2400                                                                          - ACGACGTTTA CGACAATAAC GTTGACAAAG ACCTCACAGG AGCAAAGAAT TA - #TTGGGTAA       2460                                                                          - ACCCCGCGAT CGGCAACCTA ACGACTAAAA TCGACCTGAT AAACGTCGCA AA - #AACCGAAT       2520                                                                          - ATCTATACAA AGATTTGAAT TCAGTTTAGG TTACTTTCGT GATATGGGTC AT - #TGATGTTT       2580                                                                          - TTTAAATTTT CTTTCACAAT TCTTACCATT AAGACCTTTA TTATCACTAC CT - #CTTCTTTT       2640                                                                          - ACTACTATTA TAGCTACTTC TTTTCGAAGT TGTTCGATTT CTTTACTAAT TT - #ATATACAG       2700                                                                          - AGATCAAAGA CGATACCTTG TCGTCCTTGT ATTTCGATAA TTTTTTTTAT CA - #CCGGGACG       2760                                                                          - GGAAGATCGT TCAGTGTAAT GTTTGGATAG AGAATTTGTA GCACCAGGTT TT - #ATGTTTGC       2820                                                                          - AAACTTTTTA CATTTACTTT TACTTTCATT TCAAATTGGG CCCATGGCTC GA - #GCTTAAGA       2880                                                                          - AAAATAACTA ATTGATCAGT TTACTCATAT ATATTAACTT TTTCATTTTA TA - #TTTAGTAT       2940                                                                          #     2951                                                                    - (2) INFORMATION FOR SEQ ID NO:106:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 879 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                               - Met Phe Ser Leu Tyr Leu Tyr Ile Phe Phe Il - #e Ile Tyr Thr Leu Ile         #                15                                                           - Ile Cys Asp Pro Thr Thr Pro Glu Ser Thr Il - #e Asn Pro Leu Asn His         #            30                                                               - His Asn Leu Ser Thr Pro Lys Pro Thr Ser As - #p Asp Ile Arg Glu Ile         #        45                                                                   - Leu Arg Glu Ser Gln Ile Glu Ser Asp Asp Th - #r Ser Thr Phe Tyr Met         #    60                                                                       - Cys Pro Pro Pro Ser Gly Ser Thr Leu Val Ar - #g Leu Glu Pro Pro Arg         #80                                                                           - Ala Cys Pro Asn Tyr Lys Leu Gly Lys Asn Ph - #e Thr Glu Gly Ile Ala         #                95                                                           - Val Ile Phe Lys Glu Asn Ile Ser Pro Tyr Ly - #s Phe Lys Ala Asn Ile         #           110                                                               - Tyr Tyr Lys Asn Ile Ile Ile Thr Thr Val Tr - #p Ser Gly Ser Thr Tyr         #       125                                                                   - Ala Val Ile Thr Asn Arg Tyr Thr Asp Arg Va - #l Pro Ile Gly Val Pro         #   140                                                                       - Glu Ile Thr Glu Leu Ile Asp Arg Arg Gly Me - #t Cys Leu Ser Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asp Tyr Ile Arg Asn Asn Tyr Glu Phe Thr Al - #a Phe Asp Lys Asp Glu         #               175                                                           - Asp Pro Arg Glu Val His Leu Lys Pro Ser Ly - #s Phe Asn Thr Pro Gly         #           190                                                               - Ser Arg Gly Trp His Thr Val Asn Asp Thr Ty - #r Thr Lys Ile Gly Gly         #       205                                                                   - Ser Gly Phe Tyr His Ser Gly Thr Ser Val As - #n Cys Ile Val Glu Glu         #   220                                                                       - Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Se - #r Phe Ala Ile Ser Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Asp Ile Ile His Met Ser Pro Phe Phe Gl - #y Leu Arg Asp Gly Ala         #               255                                                           - His Thr Glu Tyr Ile Ser Tyr Ser Thr Asp Ar - #g Phe Gln Gln Ile Glu         #           270                                                               - Gly Tyr Tyr Pro Ile Asp Leu Asp Thr Arg Le - #u Gln Leu Gly Ala Pro         #       285                                                                   - Val Ser Arg Asn Phe Leu Thr Thr Gln His Va - #l Thr Val Ala Trp Asn         #   300                                                                       - Trp Val Pro Lys Ile Arg Glu Val Cys Thr Le - #u Ala Lys Trp Arg Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Asp Glu Ile Ile Arg Asp Glu Tyr Lys Gl - #y Ser Tyr Arg Phe Thr         #               335                                                           - Ala Lys Ser Ile Ser Ala Thr Phe Ile Ser As - #p Thr Thr Gln Phe Asp         #           350                                                               - Ile Asp Arg Val Lys Leu Ser Asp Cys Ala Ly - #s Arg Glu Ala Ile Glu         #       365                                                                   - Ala Ile Asp Lys Ile Tyr Lys Lys Lys Tyr As - #n Lys Thr His Ile Gln         #   380                                                                       - Thr Gly Glu Leu Glu Thr Tyr Leu Ala Arg Gl - #y Gly Phe Ile Ile Ala         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Phe Arg Pro Met Ile Ser Asn Glu Leu Ala Ly - #s Leu Tyr Ile Asn Glu         #               415                                                           - Leu Val Arg Ser Asn Arg Thr Val Asp Leu Ly - #s Ser Leu Leu Asn Pro         #           430                                                               - Ser Val Arg Gly Gly Ala Arg Lys Arg Arg Se - #r Val Glu Glu Asn Lys         #       445                                                                   - Arg Ser Lys Arg Asn Ile Glu Gly Gly Ile Gl - #u Asn Val Asn Asn Ser         #   460                                                                       - Thr Ile Ile Lys Thr Thr Ser Ser Val His Ph - #e Ala Met Leu Gln Phe         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ala Tyr Asp His Ile Gln Ser His Val Asn Gl - #u Met Leu Ser Arg Ile         #               495                                                           - Ala Thr Ala Trp Cys Asn Leu Gln Asn Lys Gl - #u Arg Thr Leu Trp Asn         #           510                                                               - Glu Val Met Lys Leu Asn Pro Thr Ser Val Al - #a Ser Val Ala Met Asp         #       525                                                                   - Gln Arg Val Ser Ala Arg Met Leu Gly Asp Va - #l Leu Ala Val Thr Gln         #   540                                                                       - Cys Val Asn Ile Ser Gly Ser Ser Val Phe Il - #e Gln Asn Ser Met Arg         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Val Leu Gly Ser Thr Thr Thr Cys Tyr Ser Ar - #g Pro Leu Ile Ser Phe         #               575                                                           - Lys Ala Leu Glu Asn Ser Thr Asn Tyr Ile Gl - #u Gly Gln Leu Gly Glu         #           590                                                               - Asn Asn Glu Leu Leu Val Glu Arg Lys Leu Il - #e Glu Pro Cys Thr Ala         #       605                                                                   - Asn His Lys Arg Tyr Phe Lys Phe Gly Ala As - #p Tyr Val Tyr Phe Glu         #   620                                                                       - Asn Tyr Ala Tyr Val Arg Lys Val Pro Leu As - #n Glu Ile Glu Met Ile         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ser Ala Tyr Val Asp Leu Asn Ile Thr Leu Le - #u Glu Asp Arg Glu Phe         #               655                                                           - Leu Pro Leu Glu Val Tyr Thr Arg Ala Glu Le - #u Glu Asp Thr Gly Leu         #           670                                                               - Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gl - #n Leu His Ala Leu Lys         #       685                                                                   - Phe Tyr Asp Ile Asp Ser Val Val Lys Val As - #p Asn Asn Val Val Ile         #   700                                                                       - Met Arg Gly Ile Ala Asn Phe Phe Gln Gly Le - #u Gly Asp Val Gly Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Phe Gly Lys Val Val Leu Gly Ala Ala As - #n Ala Val Ile Ala Thr         #               735                                                           - Val Ser Gly Val Ser Ser Phe Leu Asn Asn Pr - #o Phe Gly Ala Leu Ala         #           750                                                               - Val Gly Leu Leu Ile Leu Ala Gly Leu Phe Al - #a Ala Phe Leu Ala Tyr         #       765                                                                   - Arg Tyr Val Ser Lys Leu Lys Ser Asn Pro Me - #t Lys Ala Leu Tyr Pro         #   780                                                                       - Val Thr Thr Lys Asn Leu Lys Glu Ser Val Ly - #s Asn Gly Asn Ser Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asn Asn Ser Asp Gly Glu Glu Asn Asp Asp As - #n Ile Asp Glu Glu Lys         #               815                                                           - Leu Gln Gln Ala Lys Glu Met Ile Lys Tyr Me - #t Ser Leu Val Ser Ala         #           830                                                               - Met Glu Gln Gln Glu His Lys Ala Ile Lys Ly - #s Asn Ser Gly Pro Ala         #       845                                                                   - Leu Leu Ala Ser His Ile Thr Asn Leu Ser Le - #u Lys His Arg Gly Pro         #   860                                                                       - Lys Tyr Lys Arg Leu Lys Asn Val Asn Glu As - #n Glu Ser Lys Val             865                 8 - #70                 8 - #75                           - (2) INFORMATION FOR SEQ ID NO:107:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1615 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                               - GAGCTCGCGG CCGCCTATCA AAAGTCTTAA TGAGTTAGGT GTAGATAGTA TA - #GATATTAC         60                                                                          - TACAAAGGTA TTCATATTTC CTATCAATTC TAAAGTAGAT GATATTAATA AC - #TCAAAGAT        120                                                                          - GATGATAGTA GATAATAGAT ACGCTCATAT AATGACTGCA AATTTGGACG GT - #TCACATTT        180                                                                          - TAATCATCAC GCGTTCATAA GTTTCAACTG CATAGATCAA AATCTCACTA AA - #AAGATAGC        240                                                                          - CGATGTATTT GAGAGAGATT GGACATCTAA CTACGCTAAA GAAATTACAG TT - #ATAAATAA        300                                                                          - TACATAATGG ATTTTGTTAT CATCAGTTAT ATTTAACATA AGTACAATAA AA - #AGTATTAA        360                                                                          - ATAAAAATAC TTACTTACGA AAAAATGACT AATTAGCTAT AAAAACCCGG GC - #TGCAGCTC        420                                                                          - GAGGAATTCT TTTTATTGAT TAACTAGTCA AATGAGTATA TATAATTGAA AA - #AGTAAAAT        480                                                                          - ATAAATCATA TAATAATGAA ACGAAATATC AGTAATAGAC AGGAACTGGC AG - #ATTCTTCT        540                                                                          - TCTAATGAAG TAAGTACTGC TAAATCTCCA AAATTAGATA AAAATGATAC AG - #CAAATACA        600                                                                          - GCTTCATTCA ACGAATTACC TTTTAATTTT TTCAGACACA CCTTATTACA AA - #CTAACTAA        660                                                                          - GTCAGATGAT GAGAAAGTAA ATATAAATTT AACTTATGGG TATAATATAA TA - #AAGATTCA        720                                                                          - TGATATTAAT AATTTACTTA ACGATGTTAA TAGACTTATT CCATCAACCC CT - #TCAAACCT        780                                                                          - TTCTGGATAT TATAAAATAC CAGTTAATGA TATTAAAATA GATTGTTTAA GA - #GATGTAAA        840                                                                          - TAATTATTTG GAGGTAAAGG ATATAAAATT AGTCTATCTT TCACATGGAA AT - #GAATTACC        900                                                                          - TAATATTAAT AATTATGATA GGAATTTTTT AGGATTTACA GCTGTTATAT GT - #ATCAACAA        960                                                                          - TACAGGCAGA TCTATGGTTA TGGTAAAACA CTGTAACGGG AAGCAGCATT CT - #ATGGTAAC       1020                                                                          - TGGCCTATGT TTAATAGCCA GATCATTTTA CTCTATAAAC ATTTTACCAC AA - #ATAATAGG       1080                                                                          - ATCCTCTAGA TATTTAATAT TATATCTAAC AACAACAAAA AAATTTAACG AT - #GTATGGCC       1140                                                                          - AGAAGTATTT TCTACTAATA AAGATAAAGA TAGTCTATCT TATCTACAAG AT - #ATGAAAGA       1200                                                                          - AGATAATCAT TTAGTAGTAG CTACTAATAT GGAAAGAAAT GTATACAAAA AC - #GTGGAAGC       1260                                                                          - TTTTATATTA AATAGCATAT TACTAGAAGA TTTAAAATCT AGACTTAGTA TA - #ACAAAACA       1320                                                                          - GTTAAATGCC AATATCGATT CTATATTTCA TCATAACAGT AGTACATTAA TC - #AGTGATAT       1380                                                                          - ACTGAAACGA TCTACAGACT CAACTATGCA AGGAATAAGC AATATGCCAA TT - #ATGTCTAA       1440                                                                          - TATTTTAACT TTAGAACTAA AACGTTCTAC CAATACTAAA AATAGGATAC GT - #GATAGGCT       1500                                                                          - GTTAAAAGCT GCAATAAATA GTAAGGATGT AGAAGAAATA CTTTGTTCTA TA - #CCTTCGGA       1560                                                                          - GGAAAGAACT TTAGAACAAC TTAAGTTTAA TCAAACTTGT ATTTATGAAG GT - #ACC            1615                                                                          - (2) INFORMATION FOR SEQ ID NO:108:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1615 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                               - CTCGAGCGCC GGCGGATAGT TTTCAGAATT ACTCAATCCA CATCTATCAT AT - #CTATAATG         60                                                                          - ATGTTTCCAT AAGTATAAAG GATAGTTAAG ATTTCATCTA CTATAATTAT TG - #AGTTTCTA        120                                                                          - CTACTATCAT CTATTATCTA TGCGAGTATA TTACTGACGT TTAAACCTGC CA - #AGTGTAAA        180                                                                          - ATTAGTAGTG CGCAAGTATT CAAAGTTGAC GTATCTAGTT TTAGAGTGAT TT - #TTCTATCG        240                                                                          - GCTACATAAA CTCTCTCTAA CCTGTAGATT GATGCGATTT CTTTAATGTC AA - #TATTTATT        300                                                                          - ATGTATTACC TAAAACAATA GTAGTCAATA TAAATTGTAT TCATGTTATT TT - #TCATAATT        360                                                                          - TATTTTTATG AATGAATGCT TTTTTACTGA TTAATCGATA TTTTTGGGCC CG - #ACGTCGAG        420                                                                          - CTCCTTAAGA AAAATAACTA ATTGATCAGT TTACTCATAT ATATTAACTT TT - #TCATTTTA        480                                                                          - TATTTAGTAT ATTATTACTT TGCTTTATAG TCATTATCTG TCCTTGACCG TC - #TAAGAAGA        540                                                                          - AGATTACTTC ATTCATGACG ATTTAGAGGT TTTAATCTAT TTTTACTATG TC - #GTTTATGT        600                                                                          - CGAAGTAAGT TGCTTAATGG AAAATTAAAA AAGTCTGTGT GGAATAATGT TT - #GATTGATT        660                                                                          - CAGTCTACTA CTCTTTCATT TATATTTAAA TTGAATACCC ATATTATATT AT - #TTCTAAGT        720                                                                          - ACTATAATTA TTAAATGAAT TGCTACAATT ATCTGAATAA GGTAGTTGGG GA - #AGTTTGGA        780                                                                          - AAGACCTATA ATATTTTATG GTCAATTACT ATAATTTTAT CTAACAAATT CT - #CTACATTT        840                                                                          - ATTAATAAAC CTCCATTTCC TATATTTTAA TCAGATAGAA AGTGTACCTT TA - #CTTAATGG        900                                                                          - ATTATAATTA TTAATACTAT CCTTAAAAAA TCCTAAATGT CGACAATATA CA - #TAGTTGTT        960                                                                          - ATGTCCGTCT AGATACCAAT ACCATTTTGT GACATTGCCC TTCGTCGTAA GA - #TACCATTG       1020                                                                          - ACCGGATACA AATTATCGGT CTAGTAAAAT GAGATATTTG TAAAATGGTG TT - #TATTATCC       1080                                                                          - TAGGAGATCT ATAAATTATA ATATAGATTG TTGTTGTTTT TTTAAATTGC TA - #CATACCGG       1140                                                                          - TCTTCATAAA AGATGATTAT TTCTATTTCT ATCAGATAGA ATAGATGTTC TA - #TACTTTCT       1200                                                                          - TCTATTAGTA AATCATCATC GATGATTATA CCTTTCTTTA CATATGTTTT TG - #CACCTTCG       1260                                                                          - AAAATATAAT TTATCGTATA ATGATCTTCT AAATTTTAGA TCTGAATCAT AT - #TGTTTTGT       1320                                                                          - CAATTTACGG TTATAGCTAA GATATAAAGT AGTATTGTCA TCATGTAATT AG - #TCACTATA       1380                                                                          - TGACTTTGCT AGATGTCTGA GTTGATACGT TCCTTATTCG TTATACGGTT AA - #TACAGATT       1440                                                                          - ATAAAATTGA AATCTTGATT TTGCAAGATG GTTATGATTT TTATCCTATG CA - #CTATCCGA       1500                                                                          - CAATTTTCGA CGTTATTTAT CATTCCTACA TCTTCTTTAT GAAACAAGAT AT - #GGAAGCCT       1560                                                                          - CCTTTCTTGA AATCTTGTTG AATTCAAATT AGTTTGAACA TAAATACTTC CA - #TGG            1615                                                                          - (2) INFORMATION FOR SEQ ID NO:109:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 112 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                               - CGATGTTAAT AAGTATTACC ACAATAATTG GTGGAGCCAT TTTCGTTATA GT - #ATTGATTT         60                                                                          - TCATAACAGC TTTATGTTTC TATTGTTCAA AAAATAATAA GATCTAACTG CA - #                112                                                                          - (2) INFORMATION FOR SEQ ID NO:110:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 106 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                               - GTTAGATCTT ATTATTTTTT GAACAATAGA AACATAAAGC TGTTATGAAA AT - #CAATACTA         60                                                                          #                106CCA ATTATTGTGG TAATACTTAT TAACAT                          - (2) INFORMATION FOR SEQ ID NO:111:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                               # 20               AAGT                                                       - (2) INFORMATION FOR SEQ ID NO:112:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                               #21                TAAG T                                                     - (2) INFORMATION FOR SEQ ID NO:113:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 93 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                               - TTTTCGCGAT ATCCGTTAAG TTTGTATCGT AATGAGTTTT AAAAATTTCT AT - #CTAATATA         60                                                                          #         93       ATAA ACTCGATAAT AAC                                        - (2) INFORMATION FOR SEQ ID NO:114:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                               #         33       GAAA TCATTATAAA AGT                                        - (2) INFORMATION FOR SEQ ID NO:115:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                               #       35         TTAT TAGGTATACA AAATC                                      - (2) INFORMATION FOR SEQ ID NO:116:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 8 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                               #           8                                                                 - (2) INFORMATION FOR SEQ ID NO:117:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1760 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                               - AGTACAATAA AAAGTATTAA ATAAAAATAC TTACTTACGA AAAAATGACT AA - #TTAGCTAT         60                                                                          - AAAAACCCGG GAAAGGATCC TGATCCTTTT TCTGGGTAAG TAATACGTCA AG - #GAGAAAAC        120                                                                          - GAAACGATCT GTAGTTAGCG GCCAAACTCG AGGTCGACGG TATCGATAAG CT - #TGATTCTT        180                                                                          - TATTCTATAC TTAAAAAGTG AAAATAAATA CAAAGGTTCT TGAGGGTTGT GT - #TAAATTGA        240                                                                          - AAGCGAGAAA TAATCATAAA TTATTTCATT ATCGCGATAT CCGTTAAGTT TG - #TATCGTAA        300                                                                          - TGAGTTTTAA AAATTTTTAT CTAATATATG TAATTATAAT TTTTATAAAC TC - #GATAATAA        360                                                                          - CTTCGGCATC TACATCCAAA CCTTCAACAC CTACCATAAT TCCAACTTCA GC - #AAATGAAT        420                                                                          - CACCTGCTTC CATAGATACA ACTATAACAA AACCTATATC TACAGAGGCA AA - #TAATTTAA        480                                                                          - AATCAGTAAG TACCTCAATT AAACCACCTA AAAACTTAAA AAAAAAATTA CT - #TAAATCTA        540                                                                          - AATGTAGAGA TAATGTTATT TATAGGCCAT ATTTTAGTCA ATTAGAAATT AA - #CTGTACTA        600                                                                          - TAACTAAAAA GCAAAATTTA AGTAATCCTT TAATTGAGTT ATGGTTTAAA GA - #ACTTTCTA        660                                                                          - CATATAATAA AACCAATGAA AATGTTGAAA GTTTAAAAAC AGATATATCA AA - #AAATATTT        720                                                                          - TATTATTTTC GACAAAAAAT AATAGTGATA ACTTTTATAA TGATTTTTTA TT - #AGGTATAC        780                                                                          - AAAATCAACC AGTAAATTAT AAACTTTACG GTTCCCAATT TTATGATAAT GG - #AAACATAT        840                                                                          - TACTAAATAT AAAGTCGGTT GACTTTAAAA CCTCTGGAAT ATATACTTGG AA - #ACTATATA        900                                                                          - ATTCAAATAA TGAAAGTATT TTTGAAACTT TTAAAATTCA AGTATATGCA TA - #TCATTCCC        960                                                                          - CAAATGTAAA CTTAAAATCA AACCCAAGTT TATATAATGA AAACTACAGC GC - #TATTTGTA       1020                                                                          - CAATAGCAAA TTACTTTCCA TTGGAATCTA CGGAAATATT TTGGTTTAAC GA - #TGGACAAC       1080                                                                          - CTATTGATAA AAAATATATA GATGAAACTT ATAGTGTATG GATTGACGGT CT - #TATAACAC       1140                                                                          - GCACTTCAAT ATTATCCCTT CCCTTTTCCG AAGCCATGGA AAGCCCCCCC AA - #TTTGCGAT       1200                                                                          - GTAATGTTGA ATGGTATAAA AATTCAAAGG CATCAAAAAA ATTTTCAAAT AC - #CGTTATTC       1260                                                                          - CAAAAGTTTA CTATAAACCT TTTATATCTA TAAAATTTGA TAATGGTTTA GC - #TATTTGTG       1320                                                                          - ATGCTAAATG TGTTTCCCGT GAAAATAATA AATTACAATG GTTAGTTAAA GA - #TATACCTA       1380                                                                          - TAAATGGTGA TGATATTATA AGCGGCCCCT GTTTAAACCA CCCTGGTTTG GT - #CAATATTC       1440                                                                          - AAAATAAAAT AGATATATCG GATTATGATG AACCTGTTAC CTATAAATGT TC - #AATTATTG       1500                                                                          - GTTATCCAAT AATTTTTCCC AACTTTTATG ATGAAAAGGT GTTTGATGCA TC - #GGATGAAA       1560                                                                          - ATGTTAGTAA ATCGATGTTA ATAAGTATTA CCACAATAAT TGGTGGAGCC AT - #TTTTGTTA       1620                                                                          - TAGTATTGAT TTTTATAACA GCTTTATGTT TTTATTGTTC AAAAAATAAT AA - #GATCTAAC       1680                                                                          - TGCAAATTCT TTTTATTGAT TAACTAGTCA AATGAGTATA TATAATTGAA AA - #AGTAAAAT       1740                                                                          #                 176 - #0                                                    - (2) INFORMATION FOR SEQ ID NO:118:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1760 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                               - TCATGTTATT TTTCATAATT TATTTTTATG AATGAATGCT TTTTTACTGA TT - #AATCGATA         60                                                                          - TTTTTGGGCC CTTTCCTAGG ACTAGGAAAA AGACCCATTC ATTATGCAGT TC - #CTCTTTTG        120                                                                          - CTTTGCTAGA CATCAATCGC CGGTTTGAGC TCCAGCTGCC ATAGCTATTC GA - #ACTAAGAA        180                                                                          - ATAAGATATG AATTTTTCAC TTTTATTTAT GTTTCCAAGA ACTCCCAACA CA - #ATTTAACT        240                                                                          - TTCGCTCTTT ATTAGTATTT AATAAAGTAA TAGCGCTATA GGCAATTCAA AC - #ATAGCATT        300                                                                          - ACTCAAAATT TTTAAAAATA GATTATATAC ATTAATATTA AAAATATTTG AG - #CTATTATT        360                                                                          - GAAGCCGTAG ATGTAGGTTT GGAAGTTGTG GATGGTATTA AGGTTGAAGT CG - #TTTACTTA        420                                                                          - GTGGACGAAG GTATCTATGT TGATATTGTT TTGGATATAG ATGTCTCCGT TT - #ATTAAATT        480                                                                          - TTAGTCATTC ATGGAGTTAA TTTGGTGGAT TTTTGAATTT TTTTTTTAAT GA - #ATTTAGAT        540                                                                          - TTACATCTCT ATTACAATAA ATATCCGGTA TAAAATCAGT TAATCTTTAA TT - #GACATGAT        600                                                                          - ATTGATTTTT CGTTTTAAAT TCATTAGGAA ATTAACTCAA TACCAAATTT CT - #TGAAAGAT        660                                                                          - GTATATTATT TTGGTTACTT TTACAACTTT CAAATTTTTG TCTATATAGT TT - #TTTATAAA        720                                                                          - ATAATAAAAG CTGTTTTTTA TTATCACTAT TGAAAATATT ACTAAAAAAT AA - #TCCATATG        780                                                                          - TTTTAGTTGG TCATTTAATA TTTGAAATGC CAAGGGTTAA AATACTATTA CC - #TTTGTATA        840                                                                          - ATGATTTATA TTTCAGCCAA CTGAAATTTT GGAGACCTTA TATATGAACC TT - #TGATATAT        900                                                                          - TAAGTTTATT ACTTTCATAA AAACTTTGAA AATTTTAAGT TCATATACGT AT - #AGTAAGGG        960                                                                          - GTTTACATTT GAATTTTAGT TTGGGTTCAA ATATATTACT TTTGATGTCG CG - #ATAAACAT       1020                                                                          - GTTATCGTTT AATGAAAGGT AACCTTAGAT GCCTTTATAA AACCAAATTG CT - #ACCTGTTG       1080                                                                          - GATAACTATT TTTTATATAT CTACTTTGAA TATCACATAC CTAACTGCCA GA - #ATATTGTG       1140                                                                          - CGTGAAGTTA TAATAGGGAA GGGAAAAGGC TTCGGTACCT TTCGGGGGGG TT - #AAACGCTA       1200                                                                          - CATTACAACT TACCATATTT TTAAGTTTCC GTAGTTTTTT TAAAAGTTTA TG - #GCAATAAG       1260                                                                          - GTTTTCAAAT GATATTTGGA AAATATAGAT ATTTTAAACT ATTACCAAAT CG - #ATAAACAC       1320                                                                          - TACGATTTAC ACAAAGGGCA CTTTTATTAT TTAATGTTAC CAATCAATTT CT - #ATATGGAT       1380                                                                          - ATTTACCACT ACTATAATAT TCGCCGGGGA CAAATTTGGT GGGACCAAAC CA - #GTTATAAG       1440                                                                          - TTTTATTTTA TCTATATAGC CTAATACTAC TTGGACAATG GATATTTACA AG - #TTAATAAC       1500                                                                          - CAATAGGTTA TTAAAAAGGG TTGAAAATAC TACTTTTCCA CAAACTACGT AG - #CCTACTTT       1560                                                                          - TACAATCATT TAGCTACAAT TATTCATAAT GGTGTTATTA ACCACCTCGG TA - #AAAACAAT       1620                                                                          - ATCATAACTA AAAATATTGT CGAAATACAA AAATAACAAG TTTTTTATTA TT - #CTAGATTG       1680                                                                          - ACGTTTAAGA AAAATAACTA ATTGATCAGT TTACTCATAT ATATTAACTT TT - #TCATTTTA       1740                                                                          #                 176 - #0                                                    - (2) INFORMATION FOR SEQ ID NO:119:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 319 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                               - Leu Leu Phe Ser Thr Lys Asn Asn Ser Asp As - #n Phe Tyr Asn Asp Phe         #                15                                                           - Leu Leu Gly Ile Gln Asn Gln Pro Val Asn Ty - #r Lys Leu Tyr Gly Ser         #            30                                                               - Gln Phe Tyr Asp Asn Gly Asn Ile Leu Leu As - #n Ile Lys Ser Val Asp         #        45                                                                   - Phe Lys Thr Ser Gly Ile Tyr Thr Trp Lys Le - #u Tyr Asn Ser Asn Asn         #    60                                                                       - Glu Ser Ile Phe Glu Thr Phe Lys Ile Gln Va - #l Tyr Ala Tyr His Ser         #80                                                                           - Pro Asn Val Asn Leu Lys Ser Asn Pro Ser Le - #u Tyr Asn Glu Asn Tyr         #                95                                                           - Ser Ala Ile Cys Thr Ile Ala Asn Tyr Phe Pr - #o Leu Glu Ser Thr Glu         #           110                                                               - Ile Phe Trp Phe Asn Asp Gly Gln Pro Ile As - #p Lys Lys Tyr Ile Asp         #       125                                                                   - Glu Thr Tyr Ser Val Trp Ile Asp Gly Leu Il - #e Thr Arg Thr Ser Ile         #   140                                                                       - Leu Ser Leu Pro Phe Ser Glu Ala Met Glu Se - #r Pro Pro Asn Leu Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Cys Asn Val Glu Trp Tyr Lys Asn Ser Lys Al - #a Ser Lys Lys Phe Ser         #               175                                                           - Asn Thr Val Ile Pro Lys Val Tyr Tyr Lys Pr - #o Phe Ile Ser Ile Lys         #           190                                                               - Phe Asp Asn Gly Leu Ala Ile Cys Asp Ala Ly - #s Cys Val Ser Arg Glu         #       205                                                                   - Asn Asn Lys Leu Gln Trp Leu Val Lys Asp Il - #e Pro Ile Asn Gly Asp         #   220                                                                       - Asp Ile Ile Ser Gly Pro Cys Leu Asn His Pr - #o Gly Leu Val Asn Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gln Asn Lys Ile Asp Ile Ser Asp Tyr Asp Gl - #u Pro Val Thr Tyr Lys         #               255                                                           - Cys Ser Ile Ile Gly Tyr Pro Ile Ile Phe Pr - #o Asn Phe Tyr Asp Glu         #           270                                                               - Lys Val Phe Asp Ala Ser Asp Glu Asn Val Se - #r Lys Ser Met Leu Ile         #       285                                                                   - Ser Ile Thr Thr Ile Ile Gly Gly Ala Ile Ph - #e Val Ile Val Leu Ile         #   300                                                                       - Phe Ile Thr Ala Leu Cys Phe Tyr Cys Ser Ly - #s Asn Asn Lys Ile             305                 3 - #10                 3 - #15                           - (2) INFORMATION FOR SEQ ID NO:120:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                               - CGATATCCGT TAAGTTTGTA TCGTAATGAT TAAACTTCTA TTTATCTTAT TT - #TATTTTAA         60                                                                          #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:121:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 65 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                               - TTGGGTTAAA ATAAAATAAG ATAAATAGAA GTTTAATCAT TACGATACAA AC - #TTAACGGA         60                                                                          #            65                                                               - (2) INFORMATION FOR SEQ ID NO:122:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                               - TTGAATTCCT AAACATTTGT TGTTAATTTT TTATAATTAT TATATATTTT TT - #GTCTTTTA         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:123:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 101 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                               - TTAGATCTGT AGGAGCATCA AAAGTTGACG ATGAACTTTT CTATCTAAAT AG - #AGCTGGTC         60                                                                          #  101             ATAT TATGTTATTA AAGATTTCTA T                               - (2) INFORMATION FOR SEQ ID NO:124:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 110 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                               - TTAGATCTAG ATTCCTTACA CCATTCCATA AAAGTTGGTT CAAATTTATC TT - #CTTTAGAG         60                                                                          #             110TCGTGG TAATTGAACC ATAAAATCAG TATAGAAAAC                      - (2) INFORMATION FOR SEQ ID NO:125:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                               #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:126:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1415 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                               - AGTACAATAA AAAGTATTAA ATAAAAATAC TTACTTACGA AAAAATGACT AA - #TTAGCTAT         60                                                                          - AAAAACCCGG GAAAGGATCC TGATCCTTTT TCTGGGTAAG TAATACGTCA AG - #GAGAAAAC        120                                                                          - GAAACGATCT GTAGTTAGCG GCCAAACTCG AGGTCGACGG TATCGATAAG CT - #TGATTCTT        180                                                                          - TATTCTATAC TTAAAAAGTG AAAATAAATA CAAAGGTTCT TGAGGGTTGT GT - #TAAATTGA        240                                                                          - AAGCGAGAAA TAATCATAAA TTATTTCATT ATCGCGATAT CCGTTAAGTT TG - #TATCGTAA        300                                                                          - TGATTAAACT TCTATTTATC TTATTTTATT TTAACCCAAT AACTGGATAT AA - #ATGGGTAG        360                                                                          - ACCCTCCTCG TAGGTATAAT TACACCGTTT TAAGAATGAT TCCAGATATT CC - #AAATCCAA        420                                                                          - TGGATCCTTC TAAAAACGCT GAAGTTCGGT ATGTAACTTC TACTGACCCA TG - #TGATATGG        480                                                                          - TTGCTTTGAT TTCTAATCCA AATATAGAAT CTACAATTAA AACGATTCAA TT - #TGTGCAAA        540                                                                          - AGAAAAAATT TTACAATGCA TCTCTTAGTT GGTTTAAAGT TGGAGATGAT TG - #TACATATC        600                                                                          - CAATATATTT AATTCAATAT TTTGATTGTG ATCCTCAAAG AGAATTTGGC AT - #ATGTTTAA        660                                                                          - AAAGATCTCC AGATTTTTGG AAACCATCGT TAGTTGGTTA CACATTTTTA AC - #TGATGATG        720                                                                          - AATTGGGATT AGTTTTAGCT GCCCCCGCTC CATTTAATCA AGGTCAATAT AG - #ACGGGTTA        780                                                                          - TTCAAATTGA AAATGAAGTT TTTTATACTG ATTTTATGGT TCAATTACCA CG - #AGAAACTT        840                                                                          - GTTATTTTTC TAAAGAAGAT AAATTTGAAC CAACTTTTAT GGAATGGTGT AA - #GGAATCTA        900                                                                          - GATCTGTAGG AGCATCAAAA GTTGACGATG AACTTTTTTA TCTAAATAGA GC - #TGGTCCCC        960                                                                          - AAACCCTGCT TAAATATTAT GTTATTAAAG ATTTTTATAG ACTTAACGGT AG - #AGAACCTC       1020                                                                          - CAATAAAATT TAAAGAAGCT CTTAGATACG ATATACCATA TAAAGTGAAT GA - #TAAATTTG       1080                                                                          - ATGATGAATT ACCATCGAGG CCACATATTA GTAATACTAT TAATAAAACT AT - #TAAAGAAA       1140                                                                          - TTGTAAATCT TGAAGATTAT TTTAAAAATA CAAATGTTAT AGATACTACT AC - #CCCAACAC       1200                                                                          - CAATAAATAA TACCCCAAAA AATATAACCG TGGGAATTGT TATAATTATA TT - #AATAATAC       1260                                                                          - TATTTATAAT TGGATTTTTT GTTTATAAAA GACAAAAAAT ATATAATAAT TA - #TAAAAAAT       1320                                                                          - TAACAACAAA TGTTTAGGAA TTCTTTTTAT TGATTAACTA GTCAAATGAG TA - #TATATAAT       1380                                                                          #     1415         AAAT CATATAATAA TGAAA                                      - (2) INFORMATION FOR SEQ ID NO:127:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1415 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                               - TCATGTTATT TTTCATAATT TATTTTTATG AATGAATGCT TTTTTACTGA TT - #AATCGATA         60                                                                          - TTTTTGGGCC CTTTCCTAGG ACTAGGAAAA AGACCCATTC ATTATGCAGT TC - #CTCTTTTG        120                                                                          - CTTTGCTAGA CATCAATCGC CGGTTTGAGC TCCAGCTGCC ATAGCTATTC GA - #ACTAAGAA        180                                                                          - ATAAGATATG AATTTTTCAC TTTTATTTAT GTTTCCAAGA ACTCCCAACA CA - #ATTTAACT        240                                                                          - TTCGCTCTTT ATTAGTATTT AATAAAGTAA TAGCGCTATA GGCAATTCAA AC - #ATAGCATT        300                                                                          - ACTAATTTGA AGATAAATAG AATAAAATAA AATTGGGTTA TTGACCTATA TT - #TACCCATC        360                                                                          - TGGGAGGAGC ATCCATATTA ATGTGGCAAA ATTCTTACTA AGGTCTATAA GG - #TTTAGGTT        420                                                                          - ACCTAGGAAG ATTTTTGCGA CTTCAAGCCA TACATTGAAG ATGACTGGGT AC - #ACTATACC        480                                                                          - AACGAAACTA AAGATTAGGT TTATATCTTA GATGTTAATT TTGCTAAGTT AA - #ACACGTTT        540                                                                          - TCTTTTTTAA AATGTTACGT AGAGAATCAA CCAAATTTCA ACCTCTACTA AC - #ATGTATAG        600                                                                          - GTTATATAAA TTAAGTTATA AAACTAACAC TAGGAGTTTC TCTTAAACCG TA - #TACAAATT        660                                                                          - TTTCTAGAGG TCTAAAAACC TTTGGTAGCA ATCAACCAAT GTGTAAAAAT TG - #ACTACTAC        720                                                                          - TTAACCCTAA TCAAAATCGA CGGGGGCGAG GTAAATTAGT TCCAGTTATA TC - #TGCCCAAT        780                                                                          - AAGTTTAACT TTTACTTCAA AAAATATGAC TAAAATACCA AGTTAATGGT GC - #TCTTTGAA        840                                                                          - CAATAAAAAG ATTTCTTCTA TTTAAACTTG GTTGAAAATA CCTTACCACA TT - #CCTTAGAT        900                                                                          - CTAGACATCC TCGTAGTTTT CAACTGCTAC TTGAAAAAAT AGATTTATCT CG - #ACCAGGGG        960                                                                          - TTTGGGACGA ATTTATAATA CAATAATTTC TAAAAATATC TGAATTGCCA TC - #TCTTGGAG       1020                                                                          - GTTATTTTAA ATTTCTTCGA GAATCTATGC TATATGGTAT ATTTCACTTA CT - #ATTTAAAC       1080                                                                          - TACTACTTAA TGGTAGCTCC GGTGTATAAT CATTATGATA ATTATTTTGA TA - #ATTTCTTT       1140                                                                          - AACATTTAGA ACTTCTAATA AAATTTTTAT GTTTACAATA TCTATGATGA TG - #GGGTTGTG       1200                                                                          - GTTATTTATT ATGGGGTTTT TTATATTGGC ACCCTTAACA ATATTAATAT AA - #TTATTATG       1260                                                                          - ATAAATATTA ACCTAAAAAA CAAATATTTT CTGTTTTTTA TATATTATTA AT - #ATTTTTTA       1320                                                                          - ATTGTTGTTT ACAAATCCTT AAGAAAAATA ACTAATTGAT CAGTTTACTC AT - #ATATATTA       1380                                                                          #     1415         TTTA GTATATTATT ACTTT                                      - (2) INFORMATION FOR SEQ ID NO:128:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 345 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                               - Met Ile Lys Leu Leu Phe Ile Leu Phe Tyr Ph - #e Asn Pro Ile Thr Gly         #                15                                                           - Tyr Lys Trp Val Asp Pro Pro Arg Arg Tyr As - #n Tyr Thr Val Leu Arg         #            30                                                               - Met Ile Pro Asp Ile Pro Asn Pro Met Asp Pr - #o Ser Lys Asn Ala Glu         #        45                                                                   - Val Arg Tyr Val Thr Ser Thr Asp Pro Cys As - #p Met Val Ala Leu Ile         #    60                                                                       - Ser Asn Pro Asn Ile Glu Ser Thr Ile Lys Th - #r Ile Gln Phe Val Gln         #80                                                                           - Lys Lys Lys Phe Tyr Asn Ala Ser Leu Ser Tr - #p Phe Lys Val Gly Asp         #                95                                                           - Asp Cys Thr Tyr Pro Ile Tyr Leu Ile Gln Ty - #r Phe Asp Cys Asp Pro         #           110                                                               - Gln Arg Glu Phe Gly Ile Cys Leu Lys Arg Se - #r Pro Asp Phe Trp Lys         #       125                                                                   - Pro Ser Leu Val Gly Tyr Thr Phe Leu Thr As - #p Asp Glu Leu Gly Leu         #   140                                                                       - Val Leu Ala Ala Pro Ala Pro Phe Asn Gln Gl - #y Gln Tyr Arg Arg Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Gln Ile Glu Asn Glu Val Phe Tyr Thr As - #p Phe Met Val Gln Leu         #               175                                                           - Pro Arg Glu Thr Cys Tyr Phe Ser Lys Glu As - #p Lys Phe Glu Pro Thr         #           190                                                               - Phe Met Glu Trp Cys Lys Glu Ser Arg Ser Va - #l Gly Ala Ser Lys Val         #       205                                                                   - Asp Asp Glu Leu Phe Tyr Leu Asn Arg Ala Gl - #y Pro Gln Thr Leu Leu         #   220                                                                       - Lys Tyr Tyr Val Ile Lys Asp Phe Tyr Arg Le - #u Asn Gly Arg Glu Pro         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Ile Lys Phe Lys Glu Ala Leu Arg Tyr As - #p Ile Pro Tyr Lys Val         #               255                                                           - Asn Asp Lys Phe Asp Asp Glu Leu Pro Ser Ar - #g Pro His Ile Ser Asn         #           270                                                               - Thr Ile Asn Lys Thr Ile Lys Glu Ile Val As - #n Leu Glu Asp Tyr Phe         #       285                                                                   - Lys Asn Thr Asn Val Ile Asp Thr Thr Thr Pr - #o Thr Pro Ile Asn Asn         #   300                                                                       - Thr Pro Lys Asn Ile Thr Val Gly Ile Val Il - #e Ile Ile Leu Ile Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Phe Ile Ile Gly Phe Phe Val Tyr Lys Ar - #g Gln Lys Ile Tyr Asn         #               335                                                           - Asn Tyr Lys Lys Leu Thr Thr Asn Val                                         #           345                                                               __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid coding for canineherpesvirus gD glycoprotein and having the sequence as set forth in SEQID NO:18.
 2. The isolated nucleic acid of claim 1 which is DNA.
 3. Avector containing the isolated nucleic acid of claim
 2. 4. The vector ofclaim 3 wherein the vector is a poxvirus.
 5. The vector of claim 4wherein the poxvirus is an avipox virus or a vaccinia virus.
 6. Thevector of claim 5 wherein the poxvirus is a vaccinia virus.
 7. Thevector of claim 6 wherein the deleted genetic functions include aC7L-K1L open reading frame, or, host range functions.
 8. The vector ofclaim 7 wherein at least one additional open reading frame is deleted;and, the additional open reading frame is selected from the groupconsisting of: J2R, B13R+B14R, A26L, A56R, and I4L.
 9. The vector ofclaim 7 wherein at least one additional region is deleted; and, theadditional region is selected from the group consisting of: a thymidinekinase gene, a hemorrhagic region, an A type inclusion body region, ahemagglutinin gene, and a large subunit, ribonucleotide reductase. 10.The vector of claim 8 wherein J2R, B13R+B14R, A26L, A56R, C7L-K1L andI4L are deleted from the virus.
 11. The vector of claim 9 wherein athymidine kinase gene, a hemorrhagic region, an A type inclusion bodyregion, a hemagglutinin gene, a host range region, and a large subunit,ribonucleotide reductase are deleted from the virus.
 12. The vector ofclaim 10 which is a NYVAC recombinant virus.
 13. The vector of claim 5wherein said virus is a canarypox virus.
 14. The vector of claim 13wherein the canarypox virus is a Rentschler vaccine strain which wasattenuated through more than 200 serial passages on chick embryofibroblasts, a master seed therefrom was subjected to four successiveplaque purifications under agar, from which a plaque clone was amplifiedthrough five additional passages.
 15. A composition for inducing anantigenic or immunological response comprising a vector as claimed inany one of claims 4, 7, 12, 13 or 14 in admixture with a suitablecarrier.
 16. A method for expressing a gene product in a cell culturedin vitro comprising introducing into the cell a vector as claimed in anyone of claims 4, 7, 12 or 14.